Novel serpentine transmembrane antigens expressed in human cancers and uses thereof

ABSTRACT

Described is a novel family of cell surface serpentine transmembrane antigens. Two of the proteins in this family are exclusively or predominantly expressed in the prostate, as well as in prostate cancer, and thus members of this family have been termed “STEAP” (Six Transmembrane Epithelial Antigens of the Prostate). Four particular human STEAPs are described and characterized herein. The prototype member of the STEAP family, STEAP-1, appears to be a type IIIa membrane protein expressed predominantly in prostate cells in normal human tissues. Structurally, STEAP-1 is a 339 amino acid protein characterized by a molecular topology of six transmembrane domains and intracellular N- and C-termini, suggesting that it folds in a “serpentine” manner into three extracellular and two intracellular loops. STEAP-1 protein expression is maintained at high levels across various stages of prostate cancer. Moreover, STEAP-1 is highly over-expressed in certain other human cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/575,164, filed Oct. 7, 2009, which is adivisional of U.S. patent application Ser. No. 11/225,661, filed Sep.12, 2005, now issued as U.S. Pat. No. 7,642,054, which is a continuationof U.S. patent application Ser. No. 10/750,262, filed Dec. 31, 2003, nowissued as U.S. Pat. No. 7,166,714 on Jan. 23, 2007, which is adivisional of U.S. patent application Ser. No. 10/011,095, filed Dec. 6,2001, now issued as U.S. Pat. No. 7,053,186 on May 30, 2006, which is adivisional of U.S. patent application Ser. No. 09/323,873, filed Jun. 1,1999, now issued as U.S. Pat. No. 6,329,503 on Dec. 11, 2001, whichclaims the benefit of U.S. Provisional Application No. 60/091,183, filedJun. 30, 1998 and U.S. Provisional Application No. 60/087,520, filedJun. 1, 1998. This application relates to U.S. Provisional ApplicationNo. 60/317,840, filed Sep. 6, 2001; U.S. Provisional Application No.60/370,387, filed Apr. 5, 2002, 2002; U.S. patent application Ser. No.10/010,667, filed Dec. 6, 2001, now issued as U.S. Pat. No. 6,887,975 onMay 3, 2005; U.S. patent application Ser. No. 10/858,887, filed Jun. 1,2004, now issued as U.S. Pat. No. 7,575,749 on Aug. 18, 2009; U.S.patent application Ser. No. 11/225,661, filed Sep. 12, 2005, now issuedas U.S. Pat. No. 7,642,054 on Jan. 5, 2010; U.S. patent application Ser.No. 10/236,878, filed Sep. 6, 2002, now abandoned, U.S. patentapplication Ser. No. 10/830,899, filed Apr. 23, 2004, now issued as U.S.Pat. No. 7,494,646 on Feb. 4, 2009; and U.S. patent application Ser. No.10/861,662, filed Jun. 4, 2004, now issued as U.S. Pat. No. 7,939,503 onMay 10, 2011. The contents of the applications listed in this paragraphare fully incorporated by reference herein.

INCORPORATION OF SEQUENCE LISTING

This application includes a Sequence Listing submitted via EFS-Web as acomputer-readable form 21,778 byte file entitled “GNE-0160R1D1C1D1.txt”created on Dec. 16, 2009, which is hereby incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention described herein relates to a family of novel genes andtheir encoded proteins and tumor antigens, termed STEAPs, and todiagnostic and therapeutic methods and compositions useful in themanagement of various cancers, particularly including prostate cancer,colon cancer, bladder cancer, ovarian cancer and pancreatic cancer.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Around the world, millions of people die from cancer everyyear. In the United States alone, cancer causes the death of well over ahalf-million people each year, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the leading causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.

Generally speaking, the fundamental problem in the management of thedeadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine, still very much in its infancy, promisesto redefine the ways in which these cancers are managed. Unquestionably,there is an intensive worldwide effort aimed at the development of novelmolecular approaches to cancer diagnosis and treatment. For example,there is a great interest in identifying truly tumor-specific genes andproteins that could be used as diagnostic and prognostic markers and/ortherapeutic targets or agents. Research efforts in these areas areencouraging, and the increasing availability of useful moleculartechnologies has accelerated the acquisition of meaningful knowledgeabout cancer. Nevertheless, progress is slow and generally uneven.

Recently, there has been a particularly strong interest in identifyingcell surface tumor-specific antigens which might be useful as targetsfor various immunotherapeutic or small molecule treatment strategies. Alarge number of such cell-surface antigens have been reported, and somehave proven to be reliably associated with one or more cancers. Muchattention has been focused on the development of novel therapeuticstrategies which target these antigens. However, few truly effectiveimmunological cancer treatments have resulted.

The use of monoclonal antibodies to tumor-specific or over-expressedantigens in the treatment of solid cancers is instructive. Althoughantibody therapy has been well researched for some 20 years, only veryrecently have corresponding pharmaceuticals materialized. One example isthe humanized anti-HER2/neu monoclonal antibody, Herceptin, recentlyapproved for use in the treatment of metastatic breast cancersoverexpressing the HER2/neu receptor.

Another is the human/mouse chimeric anti-CD20/B cell lymphoma antibody,Rituxan, approved for the treatment of non-Hodgkin's lymphoma. Severalother antibodies are being evaluated for the treatment of cancer inclinical trials or in pre-clinical research, including a fully humanIgG2 monoclonal antibody specific for the epidermal growth factorreceptor (Yang et al., 1999, Cancer Res. 59: 1236). Evidently, antibodytherapy is finally emerging from a long embryonic phase. Nevertheless,there is still a very great need for new, more-specific tumor antigensfor the application of antibody and other biological therapies. Inaddition, there is a corresponding need for tumor antigens which may beuseful as markers for antibody-based diagnostic and imaging methods,hopefully leading to the development of earlier diagnosis and greaterprognostic precision.

As discussed below, the management of prostate cancer serves as a goodexample of the limited extent to which molecular biology has translatedinto real progress in the clinic. With limited exceptions, the situationis more or less the same for the other major carcinomas mentioned above.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of this disease,second only to lung cancer. Despite the magnitude of these figures,there is still no effective treatment for metastatic prostate cancer.Surgical prostatectomy, radiation therapy, hormone ablation therapy, andchemotherapy remain as the main treatment modalities. Unfortunately,these treatments are clearly ineffective for many. Moreover, thesetreatments are often associated with significant undesirableconsequences.

On the diagnostic front, the serum PSA assay has been a very usefultool. Nevertheless, the specificity and general utility of PSA is widelyregarded as lacking in several respects. Neither PSA testing, nor anyother test nor biological marker has been proven capable of reliablyidentifying early-stage disease. Similarly, there is no marker availablefor predicting the emergence of the typically fatal metastatic stage ofthe disease. Diagnosis of metastatic prostate cancer is achieved by opensurgical or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy analysis.Clearly, better imaging and other less invasive diagnostic methods offerthe promise of easing the difficulty those procedures place on apatient, as well as improving therapeutic options. However, until thereare prostate tumor markers capable of reliably identifying early-stagedisease, predicting susceptibility to metastasis, and precisely imagingtumors, the management of prostate cancer will continue to be extremelydifficult. Accordingly, more specific molecular tumor markers areclearly needed in the management of prostate cancer.

There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54: 1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252) andprostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.Acad. Sci. USA 95: 1735). PCTA-1, a novel galectin, is largely secretedinto the media of expressing cells and may hold promise as a diagnosticserum marker for prostate cancer (Su et al., 1996). PSCA, a GPI-linkedcell surface molecule, was cloned from LAPC-4 cDNA and is unique in thatit is expressed primarily in basal cells of normal prostate tissue andin cancer epithelia (Reiter et al., 1998). Vaccines for prostate cancerare also being actively explored with a variety of antigens, includingPSM and PSA.

SUMMARY OF THE INVENTION

The present invention relates to a novel family of cell surfaceserpentine transmembrane antigens. Two of the proteins in this familyare exclusively or predominantly expressed in the prostate, as well asin prostate cancer, and thus members of this family have been termed“STEAP” (Six Transmembrane Epithelial Antigen of the Prostate). Fourparticular human STEAPs are described and characterized herein. Thehuman STEAPs exhibit a high degree of structural conservation among thembut show no significant structural homology to any known human proteins.

The prototype member of the STEAP family, STEAP-1, appears to be a typeMa membrane protein expressed predominantly in prostate cells in normalhuman tissues. Structurally, STEAP-1 is a 339 amino acid proteincharacterized by a molecular topology of six transmembrane domains andintracellular N- and C-termini, suggesting that it folds in a“serpentine” manner into three extracellular and two intracellularloops. STEAP-1 protein expression is maintained at high levels acrossvarious stages of prostate cancer. Moreover, STEAP-1 is highlyover-expressed in certain other human cancers. In particular, cellsurface expression of STEAP-1 has been definitively confirmed in avariety of prostate and prostate cancer cells, bladder cancer cells andcolon cancer cells. These characteristics indicate that STEAP-1 is aspecific cell-surface tumor antigen expressed at high levels inprostate, bladder, colon, and other cancers.

STEAP-2, STEAP-3 and STEAP-4 are also described herein. All arestructurally related, but show unique expression profiles. STEAP-2, likeSTEAP-1, is prostate-specific in normal human tissues and is alsoexpressed in prostate cancer. In contrast, STEAP-3 and STEAP-4 appear toshow a different restricted expression pattern.

The invention provides polynucleotides corresponding or complementary toall or part of the STEAP genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding STEAPproteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to theSTEAP genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides which hybridize to the STEAP genes, mRNAs, or toSTEAP-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding STEAPs. Recombinant DNA moleculescontaining STEAP polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of STEAP geneproducts are also provided. The invention further provides STEAPproteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to STEAP proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker, and antibodies conjugated to radionuclides, toxins or othertherapeutic compositions. The invention further provides methods fordetecting the presence of STEAP polynucleotides and proteins in variousbiological samples, as well as methods for identifying cells thatexpress a STEAP. The invention further provides various therapeuticcompositions and strategies for treating prostate cancer, includingparticularly, antibody, vaccine and small molecule therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. STEAP-1 structure. 1A-1-1A-2: Nucleotide and deduced amino acidsequences of STEAP-1 (8P1B4) clone 10 cDNA (SEQ ID NOS. 1 and 2,respectively). The start Methionine is indicated in bold at amino acidresidue position 1 and six putative transmembrane domains are indicatedin bold and are underlined. 1B: Schematic representation of STEAP-1transmembrane orientation; amino acid residues bordering the predictedextracellular domains are indicated and correspond to the numberingscheme of FIG. 1A. 1C: G/C rich 5′ non-coding sequence of the STEAP-1gene (SEQ ID NO:3) as determined by overlapping sequences of clone 10and clone 3.

FIG. 2. Predominant expression of STEAP-1 in prostate tissue. Firststrand cDNA was prepared from 16 normal tissues, the LAPC xenografts(4AD, 4AI and 9AD) and HeLa cells. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersderived from STEAP-1 (8P1D4) cDNA (FIG. 1A), shows predominantexpression of STEAP-1 in normal prostate and the LAPC xenografts. Thefollowing primers were used to amplify STEAP-1:

8P1D4.1 5′ ACTTTGTTGATGACCAGGATTGGA 3′ (SEQ ID NO: 4) 8P1D4.2 5′CAGAACTTCAGCACACACAGGAAC 3′ (SEQ ID NO: 5)

FIG. 3. Northern blot analyses of STEAP-1 expression in various normalhuman tissues and prostate cancer xenografts, showing predominantexpression of STEAP-1 in prostate tissue. FIG. 3A: Two multiple tissuenorthern blots (Clontech) were probed with a full length STEAP cDNAclone 10 (FIG. 1A; SEQ ID NO: 1). Size standards in kilobases (kb) areindicated on the side. Each lane contains 2 μg of mRNA that wasnormalized by using a β-actin probe. FIG. 3B: Multiple tissue RNA dotblot (Clontech, Human Master Blot cat #7770-1) probed with STEAP-1 cDNAclone 10 (FIG. 1A; SEQ ID NO: 1), showing approximately five-foldgreater expression in prostate relative to other tissues withsignificant detectable expression.

FIG. 4A-4B. Nucleotide sequence of STEAP-1 GTH9 clone (SEQ ID NO: 6)corresponding to the 4 kb message on northern blots (FIG. 3A). Thesequence contains an intron of 2399 base pairs relative to the STEAP-1clone 10 sequence of FIG. 1A; coding regions are nucleotides 96-857 and3257-3510 (indicated in bold). The start ATG is in bold and underlined,the STOP codon is in bold and underlined, and the intron-exon boundariesare underlined.

FIG. 5. Expression of STEAP-1 in prostate and multiple cancer cell linesand prostate cancer xenografts. Xenograft and cell line filters wereprepared with 10 μg of total RNA per lane. The blots were analyzed usingthe STEAP-1 clone 10 as probe. All RNA samples were normalized byethidium bromide staining and subsequent analysis with a β-actin probe.FIG. 5A: Expression in various cancer cell lines and xenografts andprostate. Lanes as follows: (1) PrEC cells, (2) normal prostate tissue,(3) LAPC-4 AD xenograft, (4) LAPC-4 AI xenograft, (5) LAPC-9 ADxenograft, (6) LAPC-9 AI xenograft, (7) LNCaP cells, (8) PC-3 cells, (9)DU145 cells, (10) PANC-1 cells, (11) BxPC-3 cells, (12) HPAC cells, (13)Capan-1 cells, (14) CACO-2 cells, (15) LOVO cells, (16) T84 cells, (17)COLO-205 cells, (18) KCL-22 cells (acute lymphocytic leukemia, ALL),(19) HT1197 cells, (20) SCABER cells, (21) UM-UC-3 cells, (22) TCCSUPcells, (23) J82 cells, (24) 5637 cells, (25) RD-ES cells (Ewing sarcoma,EWS), (26) CAMA-1 cells, (27) DU4475 cells, (28) MCF-7 cells, (29)MDA-MB-435s cells, (30) NTERA-2 cells, (31) NCCIT cells, (32) TERA-1cells, (33) TERA-2 cells, (34) A431 cells, (35) HeLa cells, (36) OV-1063cells, (37) PA-1 cells, (38) SW 626 cells, (39) CAOV-3 cells. FIG. 5B:The expression of STEAP-1 In subcutaneously (sc) grown LAPC xenograftscompared to the expression in LAPC-4 and LAPC-9 xenografts grown in thetibia (it) of mice.

FIG. 6. Western blot analysis of STEAP-1 protein expression in tissuesand multiple cell lines. Western blots of cell lysates prepared fromprostate cancer xenografts and cell lines were probed with a polyclonalanti-STEAP-1 antibody preparation (see Example 3C for details). Thesamples contain 20 μg of protein and were normalized with anti-Grb-2probing of the Western blots.

FIG. 7. Cell surface biotinylation of STEAP-1. FIG. 7A: Cell surfacebiotinylation of 293T cells transfected with vector alone or with vectorcontaining cDNA encoding 6His-tagged STEAP-1. Cell lysates wereimmunoprecipitated with specific antibodies, transferred to a membraneand probed with horseradish peroxidase-conjugated streptavidin. Lanes1-4 and 6 correspond to immunoprecipitates from lysates prepared fromSTEAP-1 expressing 293T cells. Lanes 5 and 7 are immunoprecipitates fromvector transfected cells. The immunoprecipitations were performed usingthe following antibodies: (1) sheep non-immune, (2) anti-Large Tantigen, (3) anti-CD71 (transferrin receptor), (4) anti-His, (5)anti-His, (6) anti-STEAP-1, (7) anti-STEAP-1. FIG. 7B: Prostate cancer(LNCaP, PC-3, DU145), bladder cancer (UM-UC-3, TCCSUP) and colon cancer(LOVO, COLO) cell lines were either biotinylated (+) or not (−) prior tolysis. Western blots of streptavidin-gel purified proteins were probedwith anti-STEAP-1 antibodies. Molecular weight markers are indicated inkilodaltons (kD).

FIG. 8. Immunohistochemical analysis of STEAP-1 expression usinganti-STEAP-1 polyclonal antibody. Tissues were fixed in 10% formalin andembedded in paraffin. Tissue sections were stained using anti-STEAP-1polyclonal antibodies directed towards the N-terminal peptide. Samplesinclude: (a) LNCaP cells probed in the presence of N-terminal STEAP-1peptide 1, (b) LNCaP plus non specific peptide 2, (c) normal prostatetissue, (d) grade 3 prostate carcinoma, (e) grade 4 prostate carcinoma,(f) LAPC-9 AD xenograft, (g) normal bladder, (h) normal colon. Allimages are at 400 x magnification.

FIG. 9. Partial nucleotide and deduced amino acid sequences of STEAP-2(98P4B6) clone GTA3 cDNA (SEQ ID NOS: 7 and 8, respectively). The 5′ endsequence of this clone contains an ORF of 173 amino acids.

FIG. 10. Nucleotide sequences of additional STEAP family membersidentified by searching the dbest database with the protein sequence ofSTEAP-1. In addition to STEAP-1, another three STEAP family members areindicated with their GenBank accession numbers. One of these correspondsto the gene 98P4B6 that was identified by SSH. AA5058880/SEQ ID NO.9;98P4B6 SSH/SEQ ID NO. 10; AI139607/SEQ ID NO. 11; R80991/SEQ ID NO. 12.

FIG. 11. Primary structural comparison of STEAP family proteins. FIG.11A. Amino acid sequence alignment of STEAP-1 (8P1D4 CLONE 10; SEQ IDNO:2) and STEAP-2 (98P4B6; SEQ ID NO:8) sequences. The alignment wasperformed using the SIM alignment program of the Baylor College ofMedicine Search Launcher Web site. Results show a 61.4% identity in a171 amino acid overlap; Score: 576.0; Gap frequency: 0.0%. FIG. 11B.Amino acid sequence alignment of STEAP-1 with partial ORF sequences ofSTEAP-2 and two other putative family member proteins (SEQ ID NO:35 andSEQ ID NO:36) using the PIMA 1.4 program; transmembrane domainsidentified by the SOSUI program are in bold.

FIG. 12. Predominant expression of AI139607 in placenta and prostate.First strand cDNA was prepared from 16 normal tissues. Normalization wasperformed by PCR using primers to actin and GAPDH. Semi-quantitativePCR, using primers to AI139607, shows predominant expression of AI139607in placenta and prostate after 25 cycles of amplification. The followingprimers were used to amplify AI139607:

A1139607.1 5′ TTAGGACAACTTGATCACCAGCA 3′ (SEQ ID NO: 13) A1139607.2 5′TGTCCAGTCCAAACTGGGTTATTT 3′ (SEQ ID NO: 14)

FIG. 13. Predominant expression of R80991 in liver. First strand cDNAwas prepared from 16 normal, tissues. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto R80991, shows predominant expression of R80991 in liver after 25cycles of amplification. The following primers were used to amplifyR80991:

R80991.1 5′ AGGGAGTTCAGCTTCGTTCAGTC 3′ (SEQ ID NO: 15) R80991.2 5′GGTAGAACTTGTAGCGGCTCTCCT 3′ (SEQ ID NO: 16)

FIG. 14. Predominant expression of STEAP-2 (98P4B6) in prostate tissue.First strand cDNA was prepared from 8 normal tissues, the LAPCxenografts (4AD, 4AI and 9AD) and HeLa cells. Normalization wasperformed by PCR using primers to actin and GAPDH.

Semi-quantitative PCR, using primers to 98P4B6, shows predominantexpression of 98P4B6 in normal prostate and the LAPC xenografts. Thefollowing primers were used to amplify STEAP II:

98P4B6.1 5′ GACTGAGCTGGAACTGGAATTTGT 3′ (SEQ ID NO: 17) 98P4B6.2 5′TTTGAGGAGACTTCATCTCACTGG 3′ (SEQ ID NO: 18)

FIG. 15. Lower expression of the prostate-specific STEAP-2/98P4B6 genein prostate cancer xenografts determined by Northern blot analysis.Human normal tissue filters (A and B) were obtained from CLONTECH andcontain 2 μg of mRNA per lane. Xenograft filter (C) was prepared with 10μg of total RNA per lane. The blots were analyzed using the SSH derived98P4B6 clone as probe. All RNA samples were normalized by ethidiumbromide staining.

FIG. 16. Expression of STEAP-2 in prostate and select cancer cell linesas determined by Northern blot analysis. Xenograft and cell line filterswere prepared with 10 μg total RNA per lane. The blots were analyzedusing an SSH derived 98P4B6 clone as probe. All RNA samples werenormalized by ethidium bromide staining.

FIG. 17. Chromosomal localization of STEAP family members. Thechromosomal localizations of the STEAP genes described herein weredetermined using the GeneBridge4 radiation hybrid panel (ResearchGenetics, Huntsville, Ala.). The mapping for STEAP-2 and AI139607 wasperformed using the Stanford G3 radiation hybrid panel (ResearchGenetics, Huntsville, Ala.).

FIG. 18. Schematic representation of Intron-Exon boundaries within theORF of human STEAP-1 gene. A total of 3 Introns (i) and 4 exons (e) wereidentified.

FIG. 19. Zooblot southern analysis of STEAP-1 gene in various species.Genomic DNA was prepared from several different organisms includinghuman, monkey, dog, mouse, chicken and Drosophila. Ten micrograms ofeach DNA sample was digested with EcoRI, blotted onto nitrocellulose andprobed with a STEAP-1 probe. Size standards are indicated on the side inkilobases (kb).

FIG. 20. Southern blot analysis of mouse BAC with a STEAP-1 probe. DNAwas prepared from human cells to isolate genomic DNA and from a mouseBAC clone (12P11) that contains the mouse STEAP gene. Each DNA samplewas digested with EcoRI, blotted onto nitrocellulose and probed. Eightmicrograms of genomic DNA was compared to 250 ng of mouse BAC DNA.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers which have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers which have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least 10amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C., and most preferably to stringenthybridization conditions.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative position which are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions which are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. Further details regarding amino acidsubstitutions, which are considered conservative under such criteria,are provided below.

Additional definitions are provided throughout the subsections whichfollow.

Molecular and Biochemical Features of the STEAPs

The invention relates to a novel family of proteins, termed STEAPs. FourSTEAPs are specifically described herein by way of structural, molecularand biochemical features. As is further described in the Examples whichfollow, the STEAPs have been characterized in a variety of ways. Forexample, analyses of nucleotide coding and amino acid sequences wereconducted in order to identify conserved structural elements within theSTEAP family. Extensive RT-PCR and Northern blot analyses of STEAP mRNAexpression were conducted in order to establish the range of normal andcancerous tissues expressing the various STEAP messages. Western blot,immunohistochemical and flow cytometric analyses of STEAP proteinexpression were conducted to determine protein expression profiles, cellsurface localization and gross molecular topology of STEAP.

The prototype member of the STEAP family, STEAP-1, is asix-transmembrane cell surface protein of 339 amino acids with noidentifiable homology to any known human protein. The cDNA nucleotideand deduced amino acid sequences of human STEAP-1 are shown in FIG. 1A.A gross topological schematic of the STEAP-1 protein integrated withinthe cell membrane is shown in FIG. 1B. STEAP-1 expression ispredominantly prostate-specific in normal tissues. Specifically,extensive analysis of STEAP-1 mRNA and protein expression in normalhuman tissues shows that STEAP-1 protein is predominantly expressed inprostate and, to a far smaller degree, in bladder. STEAP-1 mRNA is alsorelatively prostate specific, with only very low level expressiondetected in a few other normal tissues. In cancer, STEAP-1 mRNA andprotein is consistently expressed at high levels in prostate cancer andduring all stages of the disease. STEAP-1 is also expressed in othercancers. Specifically, STEAP-1 mRNA is expressed at very high levels inbladder, colon, pancreatic, and ovarian cancer (as well as othercancers). In addition, cell surface expression of STEAP-1 protein hasbeen established in prostate, bladder and colon cancers. Therefore,STEAP-1 has all of the hallmark characteristics of an excellenttherapeutic target for the treatment of certain cancers, includingparticularly prostate, colon and bladder carcinomas.

STEAP-2 is a highly homologous transmembrane protein encoded by adistinct gene. The STEAP-1 and STEAP-2 sequences show a high degree ofstructural conservation, particularly throughout their predictedtransmembrane domains. The partial cDNA nucleotide and deduced aminoacid sequences of STEAP-2 are shown in FIG. 9. Both the STEAP-1 andSTEAP-2 genes are located on chromosome 7, but on different arms.STEAP-2 exhibits a markedly different mRNA and protein expressionprofile relative to STEAP-1. suggesting that these two STEAP familymembers may be differentially regulated. STEAP-2 appears to be veryprostate-specific, as significant mRNA expression is not detected in avariety of normal tissues. In prostate cancer, STEAP-2 also appears tofollow a different course relative to STEAP-1, since STEAP-2 expressionis down-regulated in at least some prostate cancers. In addition,STEAP-2 expression in other non-prostate cancers tested seems generallyabsent, although high level expression of STEAP-2 (like STEAP-1) isdetected in Ewing sarcoma.

STEAP-3 and STEAP-4 appear to be closely related to both STEAP-1 andSTEAP-2 on a structural level, and both appear to be transmembraneproteins as well. STEAP-3 and STEAP-4 show unique expression profiles.STEAP-3, for example, appears to have an expression pattern which ispredominantly restricted to placenta and, to a smaller degree,expression is seen in prostate but not in other normal tissues tested.STEAP-4 seems to be expressed predominantly in liver. Neither STEAP-3nor STEAP-4 appear to be expressed in prostate cancer xenografts whichexhibit high level STEAP-1 and STEAP-2 expression.

Three of the four STEAPs described herein map to human chromosome 7(STEAP-1, -2 and 3). Interestingly, STEAP-1 maps within 7p22 (7p22.3), alarge region of allelic gain reported for both primary and recurrentprostate cancers (Visakorpi et al., 1995 Cancer Res. 55: 342, Nupponenet al., 1998 American J. Pathol. 153: 141), suggesting thatup-regulation of STEAP-1 in cancer might include genomic mechanisms.

The function of the STEAPs are not known. Other cell surface moleculesthat contain six transmembrane domains include ion channels (Dolly andParcej, 1996 J Bioenerg Biomembr 28:231) and water channels oraquaporins (Reizer et al., 1993 Crit Rev Biochem Mol Biol 28:235).Structural studies show that both types of molecules assemble intotetrameric complexes to form functional channels (Christie, 1995, ClinExp Pharmacol Physiol 22:944, Walz et al., 1997 Nature 387:624, Cheng etal., 1997 Nature 387:627). Immunohistochemical staining of STEAP-1 inthe prostate gland seems to be concentrated at the cell-cell boundaries,with less staining detected at the luminal side. This may suggest a rolefor STEAP-1 in tight-junctions, gap-junctions or cell adhesion. In orderto test these possibilities, xenopus oocytes (or other cells) expressingSTEAP may being analyzed using voltage-clamp and patch-clamp experimentsto determine if STEAP functions as an ion-channel. Oocyte cell volumemay also be measured to determine if STEAP exhibits water channelproperties. If STEAPs function as channel or gap-junction proteins, theymay serve as excellent targets for inhibition using, for example,antibodies, small molecules, and polynucleotides capable of inhibitingexpression or function. The restricted expression pattern in normaltissue, and the high levels of expression in cancer tissue suggest thatinterfering with STEAP function may selectively kill cancer cells.

Since the STEAP gene family is predominantly expressed in epithelialtissue, it seems possible that the STEAP proteins function as ionchannels or gap-junction proteins in epithelial cell function. Ionchannels have been implicated in proliferation and invasiveness ofprostate cancer cells (Lalani et al., 1997, Cancer Metastasis Rev16:29). Both rat and human prostate cancer cells contain sub-populationof cells with higher and lower expression levels of sodium channels.Higher levels of sodium channel expression correlate with moreaggressive invasiveness in vitro (Smith et al., 1998, FEBS Lett.423:19). Similarly, it has been shown that a specific blockade of sodiumchannels inhibits the invasiveness of PC-3 cells in vitro (Laniado etal., 1997, Am. J. Pathol. 150:1213), while specific inhibition ofpotassium channels in LNCaP cells inhibited cell proliferation (Skrymaet al., 1997, Prostate 33:112). These reports suggest a role for ionchannels in prostate cancer and also demonstrate that small moleculesthat inhibit ion channel function may interfere with prostate cancerproliferation.

STEAP Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a STEAP gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a STEAP protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a STEAP gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides which hybridize to a STEAP gene,mRNA, or to a STEAP-encoding polynucleotide (collectively, “STEAPpolynucleotides”). As used herein, STEAP genes and proteins are meant toinclude the STEAP-1 and STEAP-2 genes and proteins, the genes andproteins corresponding to GeneBank Accession numbers AI139607 and R80991(STEAP-3 and STEAP-4, respectively), and the genes and proteinscorresponding to other STEAP proteins and structurally similar variantsof the foregoing. Such other STEAP proteins and variants will generallyhave coding sequences which are highly homologous to the STEAP-1 and/orSTEAP-2 coding sequences, and preferably will share at least about 50%amino acid identity and at least about 60% amino acid homology (usingBLAST criteria), more preferably sharing 70% or greater homology (usingBLAST criteria).

The STEAP family member gene sequences described herein encode STEAPproteins sharing unique highly conserved amino acid sequence domainswhich distinguish them from other proteins. Proteins which include oneor more of these unique highly conserved domains may be related to theSTEAP family members or may represent new STEAP proteins. Referring toFIG. 11A, which is an amino acid sequence alignment of the full STEAP-1and partial STEAP-2 protein sequences, the STEAP-1 and STEAP-2 sequencesshare 61% identity and 79% homology, with particularly close sequenceconservation in the predicted transmembrane domains. Referring to FIG.11B, which is an amino acid alignment of the available structures of thefour STEAP family members, very close conservation is apparent in theoverlapping regions, particularly in the fourth and fifth transmembranedomains and the predicted intracellular loop between them. Amino acidsequence comparisons show that (1) STEAP-2 and STEAP-3 are 50% identicaland 69% homologous in their overlapping sequences; (2) STEAP-2 andSTEAP-4 are 56% identical and 87% homologous in their overlappingsequences; (3) STEAP-3 and STEAP-1 are 37% identical and 63% homologousin their overlapping sequences; (4) STEAP-3 and STEAP-4 are 38%identical and 57% homologous in their overlapping sequences; and (5)STEAP 4 and STEAP-1 are 42% identical and 65% homologous in theiroverlapping sequences.

A STEAP polynucleotide may comprise a polynucleotide having thenucleotide sequence of human STEAP-1 as shown in FIG. 1A (SEQ ID NO. 1)or the nucleotide sequence of human STEAP-2 as shown in FIG. 9 (SEQ IDNO: 7), a sequence complementary to either of the foregoing, or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucelotide which encodes the human STEAP-1 protein aminoacid sequence as shown in FIG. 1A (SEQ ID NO. 2) or which encodes thehuman STEAP-2 protein amino acid sequence as shown in FIG. 9 (SEQ ID NO:8), a sequence complementary to either of the foregoing, or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide which is capable of hybridizing understringent hybridization conditions to the human STEAP-1 cDNA shown inFIG. 1A (SEQ ID NO. 1) or to a polynucleotide fragment thereof. Anotherembodiment comprises a polynucleotide which is capable of hybridizingunder stringent hybridization conditions to the human STEAP-2 cDNA shownin FIG. 9 (SEQ ID NO. 7) or to a polynucleotide fragment thereof.

Specifically contemplated are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone or including alternative bases, whether derivedfrom natural sources or synthesized. For example, antisense moleculescan be RNAs or other molecules, including peptide nucleic acids (PNAs)or non-nucleic acid molecules such as phosphorothioate derivatives, thatspecifically bind DNA or RNA in a base pair-dependent manner. A skilledartisan can readily obtain these classes of nucleic acid molecules usingthe STEAP polynucleotides and polynucleotide sequences disclosed herein.

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a STEAP polynucleotide in a sample and as ameans for detecting a cell expressing a STEAP protein. Examples of suchprobes include polynucleotides comprising all or part of the humanSTEAP-1 cDNA sequence shown in FIG. 1A (SEQ ID NO. 1) andpolynucleotides comprising all or part of the human STEAP-2 cDNAsequence shown in FIG. 9 (SEQ ID NO. 7). Examples of primer pairscapable of specifically amplifying STEAP mRNAs are also described in theExamples which follow. As will be understood by the skilled artisan, agreat many different primers and probes may be prepared based on thesequences provided in herein and used effectively to amplify and/ordetect a STEAP mRNA or an mRNA encoding a particular STEAP family member(e.g., STEAP-1).

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides whichcorrespond or are complementary to genes other than the STEAP gene orwhich encode polypeptides other than STEAP gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated STEAP polynucleotide.

The STEAP polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the STEAP gene(s), mRNA(s), orfragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of STEAP polypeptides; as tools for modulatingor inhibiting the expression of the STEAP gene(s) and/or translation ofthe STEAP transcript(s); and as therapeutic agents.

Methods for Isolating STEAP-Encoding Nucleic Acid Molecules

The STEAP cDNA sequences described herein enable the isolation of otherpolynucleotides encoding STEAP gene product(s), as well as the isolationof polynucleotides encoding STEAP gene product homologues, alternativelyspliced isoforms, allelic variants, and mutant forms of the STEAP geneproduct. Various molecular cloning methods that can be employed toisolate full length cDNAs encoding a STEAP gene are well known (See, forexample, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2dedition, Cold Spring Harbor Press, New York, 1989; Current Protocols inMolecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). Forexample, lambda phage cloning methodologies may be convenientlyemployed, using commercially available cloning systems (e.g., Lambda ZAPExpress, Stratagene). Phage clones containing STEAP gene cDNAs may beidentified by probing with a labeled STEAP cDNA or a fragment thereof.For example, in one embodiment, the STEAP-1 cDNA (FIG. 1A) or a portionthereof can be synthesized and used as a probe to retrieve overlappingand full length cDNAs corresponding to a STEAP gene. Similarly, theSTEAP-2 cDNA sequence may be employed. A STEAP gene may be isolated byscreening genomic DNA libraries, bacterial artificial chromosomelibraries (BACs), yeast artificial chromosome libraries (YACs), and thelike, with STEAP DNA probes or primers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga STEAP polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YACs, BACs, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, for example, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a STEAP polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 cell). Examples of suitablemammalian cells include various prostate cancer cell lines such LnCaP,PC-3, DU145, LAPC-4, TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of a STEAP may be used to generate STEAP proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

A wide range of host-vector systems suitable for the expression of STEAPproteins or fragments thereof are available, see for example, Sambrooket al., 1989, supra; Current Protocols in Molecular Biology, 1995,supra). Preferred vectors for mammalian expression include but are notlimited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vectorpSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expressionvectors, STEAP may be preferably expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-I,3T3, PC-3, LNCaP and TsuPr1. The host-vector systems of the inventionare useful for the production of a STEAP protein or fragment thereof.Such host-vector systems may be employed to study the functionalproperties of STEAP and STEAP mutations.

Proteins encoded by the STEAP genes, or by fragments thereof, will havea variety of uses, including but not limited to generating antibodiesand in methods for identifying ligands and other agents and cellularconstituents that bind to a STEAP gene product. Antibodies raisedagainst a STEAP protein or fragment thereof may be useful in diagnosticand prognostic assays, imaging methodologies (including, particularly,cancer imaging), and therapeutic methods in the management of humancancers characterized by expression of a STEAP protein, such asprostate, colon, breast, cervical and bladder carcinomas, ovariancancers, testicular cancers and pancreatic cancers. Variousimmunological assays useful for the detection of STEAP proteins arecontemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting prostate cells(e.g., in radioscintigraphic imaging methods). STEAP proteins may alsobe particularly useful in generating cancer vaccines, as furtherdescribed below.

STEAP Proteins

Another aspect of the present invention provides various STEAP proteinsand polypeptide fragments thereof. As used herein, a STEAP proteinrefers to a protein that has or includes the amino acid sequence ofhuman STEAP-1 as provided in FIG. 1A (SEQ ID NO. 2), human STEAP-2 asprovided in FIG. 9 (SEQ ID NO. 8), the amino acid sequence of othermammalian STEAP homologues and variants, as well as allelic variants andconservative substitution mutants of these proteins that have STEAPbiological activity.

The STEAP proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants and homologs that can be isolated/generated andcharacterized without undue experimentation following the methodsoutlined below. Fusion proteins which combine parts of different STEAPproteins or fragments thereof, as well as fusion proteins of a STEAPprotein and a heterologous polypeptide are also included. Such STEAPproteins will be collectively referred to as the STEAP proteins, theproteins of the invention, or STEAP. As used herein, the term “STEAPpolypeptide” refers to a polypeptide fragment or a STEAP protein of atleast 10 amino acids, preferably at least 15 amino acids.

A specific embodiment of a STEAP protein comprises a polypeptide havingthe amino acid sequence of human STEAP-1 as shown in FIG. 1A (SEQ ID NO.2). Another embodiment of a STEAP protein comprises a polypeptidecontaining the partial STEAP-2 amino acid sequence as shown in FIG. 9(SEQ ID NO. 8). Another embodiment comprises a polypeptide containingthe partial STEAP-3 amino acid sequence of (SEQ ID NO:35) shown in FIG.11B. Yet another embodiment comprises a polypeptide containing thepartial STEAP-4 amino acid sequence of (SEQ ID NO:36) shown in FIG. 11B.

In general, naturally occurring allelic variants of human STEAP willshare a high degree of structural identity and homology (e.g., 90% ormore identity). Typically, allelic variants of the STEAP proteins willcontain conservative amino acid substitutions within the STEAP sequencesdescribed herein or will contain a substitution of an amino acid from acorresponding position in a STEAP homologue. One class of STEAP allelicvariants will be proteins that share a high degree of homology with atleast a small region of a particular STEAP amino acid sequence, but willfurther contain a radical departure form the sequence, such as anon-conservative substitution, truncation, insertion or frame shift.Such alleles represent mutant STEAP proteins that typically do notperform the same biological functions or do not have all of thebiological characteristics.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pKs of these two amino acid residues arenot significant. Still other changes can be considered “conservative” inparticular environments.

STEAP proteins may be embodied in many forms preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the STEAP proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated STEAP protein. A purified STEAP proteinmolecule will be substantially free of other proteins or molecules whichimpair the binding of STEAP to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a STEAP protein include a purified STEAP protein and afunctional, soluble STEAP protein. In one form, such functional, solubleSTEAP proteins or fragments thereof retain the ability to bind antibodyor other ligand.

The invention also provides STEAP polypeptides comprising biologicallyactive fragments of the STEAP amino acid sequence, such as a polypeptidecorresponding to part of the amino acid sequences for STEAP-1 as shownin FIG. 1A (SEQ ID NO. 2), STEAP-2 as shown in FIG. 9 (SEQ ID NO: 8), orSTEAP-3 (SEQ ID NO:35)or STEAP-4 (SEQ ID NO:36), as shown in FIG. 11B.Such polypeptides of the invention exhibit properties of a STEAPprotein, such as the ability to elicit the generation of antibodieswhich specifically bind an epitope associated with a STEAP protein.Polypeptides comprising amino acid sequences which are unique to aparticular STEAP protein (relative to other STEAP proteins) may be usedto generate antibodies which will specifically react with thatparticular STEAP protein. For example, referring to the amino acidalignment of the STEAP-1 and STEAP-2 structures shown in FIG. 11A, theskilled artisan will readily appreciate that each molecule containsstretches of sequence unique to its structure. These unique stretchescan be used to generate STEAP-1 or STEAP-2 specific antibodies.

STEAP polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human STEAP proteins disclosedherein. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a polypeptide fragment of a STEAPprotein. In this regard, the STEAP-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of STEAPproteins. STEAP polypeptides are particularly useful in generating andcharacterizing domain specific antibodies (e.g., antibodies recognizingan extracellular or intracellular epitope of a STEAP protein), ingenerating STEAP family member specific antibodies (e.g., anti-STEAP-1,anti-STEAP 2 antibodies), identifying agents or cellular factors thatbind to a particular STEAP or STEAP domain, and in various therapeuticcontexts, including but not limited to cancer vaccines. STEAPpolypeptides containing particularly interesting structures can bepredicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-STEAP antibodies or in identifying cellular factors thatbind to STEAP.

STEAP Antibodies

Another aspect of the invention provides antibodies that bind to STEAPproteins and polypeptides. The most preferred antibodies willselectively bind to a STEAP protein and will not bind (or will bindweakly) to non-STEAP proteins and polypeptides. Anti-STEAP antibodiesthat are particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, an antibody fragment is defined as at leasta portion of the variable region of the immunoglobulin molecule whichbinds to its target, i.e., the antigen binding region.

For some applications, it may be desirable to generate antibodies whichspecifically react with a particular STEAP protein and/or an epitopewithin a particular structural domain. For example, preferred antibodiesuseful for cancer therapy and diagnostic imaging purposes are thosewhich react with an epitope in an extracellular region of the STEAPprotein as expressed in cancer cells. Such antibodies may be generatedby using the STEAP proteins described herein, or using peptides derivedfrom predicted extracellular domains thereof, as an immunogen. In thisregard, with reference to the STEAP-1 protein topological schematicshown in FIG. 1B, regions in the extracellular loops between theindicated transmembrane domains may be selected as used to designappropriate immunogens for raising extracellular-specific antibodies.

STEAP antibodies of the invention may be particularly useful in prostatecancer therapeutic strategies, diagnostic and prognostic assays, andimaging methodologies. The invention provides various immunologicalassays useful for the detection and quantification of STEAP and mutantSTEAP proteins and polypeptides. Such assays generally comprise one ormore STEAP antibodies capable of recognizing and binding a STEAP ormutant STEAP protein, as appropriate, and may be performed withinvarious immunological assay formats well known in the art, including butnot limited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer are also provided by the invention,including but limited to radioscintigraphic imaging methods usinglabeled STEAP antibodies. Such assays may be clinically useful in thedetection, monitoring, and prognosis of prostate cancer, particularlyadvanced prostate cancer.

STEAP antibodies may also be used in methods for purifying STEAP andmutant STEAP proteins and polypeptides and for isolating STEAPhomologues and related molecules. For example, in one embodiment, themethod of purifying a STEAP protein comprises incubating a STEAPantibody, which has been coupled to a solid matrix, with a lysate orother solution containing STEAP under conditions which permit the STEAPantibody to bind to STEAP; washing the solid matrix to eliminateimpurities; and eluting the STEAP from the coupled antibody. Other usesof the STEAP antibodies of the invention include generatinganti-idiotypic antibodies that mimic the STEAP protein.

STEAP antibodies may also be used therapeutically by, for example,modulating or inhibiting the biological activity of a STEAP protein ortargeting and destroying prostate cancer cells expressing a STEAPprotein. Antibody therapy of prostate and other cancers is morespecifically described in a separate subsection below.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a STEAP protein, peptide, or fragment, in isolatedor immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press,Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring HarborPress, NY (1989)). In addition, fusion proteins of STEAP may also beused, such as a STEAP GST-fusion protein. In a particular embodiment, aGST fusion protein comprising all or most of the open reading frameamino acid sequence of FIG. 1A may be produced and used as an immunogento generate appropriate antibodies. Cells expressing or overexpressingSTEAP may also be used for immunizations. Similarly, any cell engineeredto express STEAP may be used. Such strategies may result in theproduction of monoclonal antibodies with enhanced capacities forrecognizing endogenous STEAP. Another useful immunogen comprises STEAPproteins linked to the plasma membrane of sheep red blood cells.

The amino acid sequence of STEAP as shown in FIG. 1A (SEQ ID NO. 2) maybe used to select specific regions of the STEAP protein for generatingantibodies. For example, hydrophobicity and hydrophilicity analyses ofthe STEAP amino acid sequence may be used to identify hydrophilicregions in the STEAP structure. Regions of the STEAP protein that showimmunogenic structure, as well as other regions and domains, can readilybe identified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. For the generation of antibodies whichspecifically recognize a mutant STEAP protein, amino acid sequencesunique to the mutant (relative to wild type STEAP) are preferable.

Methods for preparing a protein or polypeptide for use as immunogen andfor preparing immunogenic conjugates of a protein with a carrier such asBSA, KLH, or other carrier proteins are well known in the art in somecircumstances, direct conjugation using, for example, carbodiimidereagents may be used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., may be effective.Administration of a STEAP immunogen is conducted generally by injectionover a suitable time period and with use of a suitable adjuvant, as isgenerally understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

STEAP monoclonal antibodies are preferred and may be produced by variousmeans well known in the art. For example. immortalized cell lines whichsecrete a desired monoclonal antibody may be prepared using the standardmethod of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the STEAP protein orSTEAP fragment. When the appropriate immortalized cell culture secretingthe desired antibody is identified, the cells may be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

As mentioned above, numerous STEAP polypeptides may be used asimmunogens for generating monoclonal antibodies using traditionalmethods. A particular embodiment comprises an antibody whichimmunohistochemically stains 293T cells transfected with an expressionplasmid carrying the STEAP-1 coding sequence, the transfected cellsexpressing STEAP-1 protein, but does immunohistochemically stainuntransfected 293T cells. An assay for characterizing such antibodies isprovided in Example 5 herein.

In another embodiment, STEAP-1 monoclonal antibodies may be generatedusing NIH 3T3 cells expressing STEAP-1 as an immunogen to generate mAbsthat recognize the cell surface epitopes of STEAP-1. Reactive mAbs maybe screened by cell-based ELISAs using PC-3 cells over-expressingSTEAP-1. In another specific embodiment, 3 peptides representing theextracellular regions of the STEAP-1 protein (specifically,REVIHPLATSHQQYFYKIPILV (SEQ ID NO. 19),RRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEI (SEQ ID NO. 20) and WIDIKQFVWYTPPTF(SEQ ID NO. 21) are coupled to sheep red blood cells for immunization.In another specific embodiment, recombinant STEAP-1 protein generatedwith an amino-terminal His-tag using a suitable expression system (e.g.,baculovirus expression system pBlueBac4.5, Invitrogen) is purified usinga Nickel column and used as immunogen.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the STEAP protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.Humanized or human STEAP antibodies may also be produced and arepreferred for use in therapeutic contexts. Various approaches forproducing such humanized antibodies are known, and include chimeric andCDR grafting methods; methods for producing fully human monoclonalantibodies include phage display and transgenic methods (for review, seeVaughan et al., 1998, Nature Biotechnology 16: 535-539).

Fully human STEAP monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human STEAP monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of STEAP antibodies with a STEAP protein may be establishedby a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,STEAP proteins, peptides, STEAP-expressing cells or extracts thereof.

A STEAP antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule, such as acytotoxic agent, and used for targeting the second molecule to a STEAPpositive cell (Vitetta, E. S. et al., 1993, Immunotoxin therapy, inDeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice ofOncology, 4th ed., J.B. Lippincott Co., Philadelphia, 2624-2636).Suitable detectable markers include, but are not limited to, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme.

Methods for the Detection of STEAP

Another aspect of the present invention relates to methods for detectingSTEAP polynucleotides and STEAP proteins, as well as methods foridentifying a cell which expresses STEAP.

More particularly, the invention provides assays for the detection ofSTEAP polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable STEAP polynucleotides include, for example, a STEAPgene or fragments thereof, STEAP mRNA, alternative splice variant STEAPmRNAs, and recombinant DNA or RNA molecules containing a STEAPpolynucleotide. A number of methods for amplifying and/or detecting thepresence of STEAP polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a STEAP mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a STEAPpolynucleotides as sense and antisense primers to amplify STEAP cDNAstherein; and detecting the presence of the amplified STEAP cDNA. Inanother embodiment, a method of detecting a STEAP gene in a biologicalsample comprises first isolating genomic DNA from the sample; amplifyingthe isolated genomic DNA using STEAP polynucleotides as sense andantisense primers to amplify the STEAP gene therein; and detecting thepresence of the amplified STEAP gene. Any number of appropriate senseand antisense probe combinations may be designed from the nucleotidesequences provided for STEAP-1 (FIG. 1A; SEQ ID NO. 1), STEAP-2 (FIG. 9;SEQ ID NO. 7). STEAP-3 (FIG. 10: SEQ ID NO. 11), or STEAP-4 (FIG. 10;SEQ ID NO. 12), as appropriate, and used for this purpose.

The invention also provides assays for detecting the presence of a STEAPprotein in a tissue of other biological sample such as serum, bone,prostate, and other tissues, urine, cell preparations, and the like.Methods for detecting a STEAP protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, WesternBlot analysis, molecular binding assays, ELISA, ELIFA and the like.

For example, in one embodiment, a method of detecting the presence of aSTEAP protein in a biological sample comprises first contacting thesample with a STEAP antibody, a STEAP-reactive fragment thereof, or arecombinant protein containing an antigen binding region of a STEAPantibody; and then detecting the binding of STEAP protein in the samplethereto.

Methods for identifying a cell which expresses STEAP are also provided.In one embodiment, an assay for identifying a cell which expresses aSTEAP gene comprises detecting the presence of STEAP mRNA in the cell.Methods for the detection of particular mRNAs in cells are well knownand include, for example, hybridization assays using complementary DNAprobes (such as in situ hybridization using labeled STEAP riboprobes,Northern blot and related techniques) and various nucleic acidamplification assays (such as RT-PCR using complementary primersspecific for STEAP, and other amplification type detection methods, suchas, for example, branched DNA, SISBA, TMA and the like). Alternatively,an assay for identifying a cell which expresses a STEAP gene comprisesdetecting the presence of STEAP protein in the cell or secreted by thecell. Various methods for the detection of proteins are well known inthe art and may be employed for the detection of STEAP proteins andSTEAP expressing cells.

STEAP expression analysis may also be useful as a tool for identifyingand evaluating agents which modulate STEAP gene expression. For example,STEAP-1 expression is significantly upregulated in colon, bladder,pancreatic, ovarian and other cancers. Identification of a molecule orbiological agent that could inhibit STEAP-1 over-expression may be oftherapeutic value in the treatment of cancer. Such an agent may beidentified by using a screen that quantifies STEAP expression by RT-PCR,nucleic acid hybridization or antibody binding.

Assays for Determining STEAP Expression Status

Determining the status of STEAP expression patterns in an individual maybe used to diagnose cancer and may provide prognostic information usefulin defining appropriate therapeutic options. Similarly, the expressionstatus of STEAP may provide information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining STEAP expression status and diagnosing cancers which expressSTEAP.

In one aspect, the invention provides assays useful in determining thepresence of cancer in an individual, comprising detecting a significantincrease in STEAP mRNA or protein expression in a test cell or tissuesample relative to expression levels in the corresponding normal cell ortissue. In one embodiment, the presence of STEAP-1 mRNA is evaluated intissue samples of the colon, pancreas, bladder, ovary, cervix, testis orbreast. The presence of significant STEAP-1 expression in any of thesetissues may be useful to indicate the emergence, presence and/orseverity of these cancers, since the corresponding normal tissues do notexpress STEAP-1 mRNA. In a related embodiment, STEAP-1 expression statusmay be determined at the protein level rather than at the nucleic acidlevel. For example, such a method or assay would comprise determiningthe level of STEAP-1 protein expressed by cells in a test tissue sampleand comparing the level so determined to the level of STEAP expressed ina corresponding normal sample. In one embodiment, the presence ofSTEAP-1 protein is evaluated, for example, using immunohistochemicalmethods. STEAP antibodies or binding partners capable of detecting STEAPprotein expression may be used in a variety of assay formats well knownin the art for this purpose.

Peripheral blood may be conveniently assayed for the presence of cancercells, including prostate, colon, pancreatic, bladder and ovariancancers, using RT-PCR to detect STEAP-1 expression. The presence ofRT-PCR amplifiable STEAP-1 mRNA provides an indication of the presenceof one of these types of cancer. RT-PCR detection assays for tumor cellsin peripheral blood are currently being evaluated for use in thediagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkalk et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston etal., 1995, Clin. Chem. 41: 1687-1688). RT-PCR assays are well known inthe art.

In another approach, a recently described sensitive assay for detectingand characterizing carcinoma cells in blood may be used (Racila et al.,1998, Proc. Natl. Acad. Sci. USA 95: 4589-4594). This assay combinesimmunomagnetic enrichment with multiparameter flow cytometric andimmunohistochemical analyses, and is highly sensitive for the detectionof cancer cells in blood, reportedly capable of detecting one epithelialcell in 1 ml of peripheral blood.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detectingSTEAP mRNA or STEAP protein in a tissue sample, its presence indicatingsusceptibility to cancer, wherein the degree of STEAP mRNA expressionpresent is proportional to the degree of susceptibility.

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of STEAP mRNAor STEAP protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of STEAP mRNA or STEAP proteinexpressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree ofSTEAP mRNA or STEAP protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness.

Methods for detecting and quantifying the expression of STEAP mRNA orprotein are described herein and use standard nucleic acid and proteindetection and quantification technologies well known in the art.Standard methods for the detection and quantification of STEAP mRNAinclude in situ hybridization using labeled STEAP riboprobes, Northernblot and related techniques using STEAP polynucleotide probes, RT-PCRanalysis using primers specific for STEAP, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like. In a specific embodiment, semi-quantitative RT-PCR may be usedto detect and quantify STEAP mRNA expression as described in theExamples which follow. Any number of primers capable of amplifying STEAPmay be used for this purpose, including but not limited to the variousprimer sets specifically described herein. Standard methods for thedetection and quantification of protein may be used for this purpose. Ina specific embodiment, polyclonal or monoclonal antibodies specificallyreactive with the wild-type STEAP protein may be used in animmunohistochemical assay of biopsied tissue.

Diagnostic Imaging of Human Cancers

The expression profiles of STEAP-1 and STEAP-2 indicate antibodiesspecific therefore may be particularly useful in radionuclide and otherforms of diagnostic imaging of certain cancers. For exampleimmunohistochemical analysis of STEAP-1 protein suggests that in normaltissues STEAP-1 is predominantly restricted to prostate and bladder. Thetransmembrane orientation of STEAP-1 (and presumably STEAP-2) provides atarget readily identifiable by antibodies specifically reactive withextracellular epitopes. This tissue restricted expression, and thelocalization of STEAP to the cell surface of multiple cancers makesSTEAP an ideal candidate for diagnostic imaging. Accordingly, in vivoimaging techniques may be used to image human cancers expressing a STEAPprotein.

For example, cell surface STEAP-1 protein is expressed at very highlevels in several human cancers, particularly prostate, bladder, colonand ovarian cancers, and Ewing sarcoma. Moreover, in normal tissues,STEAP-1 protein expression is largely restricted to prostate. Thus,radiolabeled antibodies specifically reactive with extracellularepitopes of STEAP-1 may be particularly useful in in vivo imaging ofsolid tumors of the foregoing cancers. Such labeled anti-STEAP-1antibodies may provide very high level sensitivities for the detectionof metastasis of these cancers.

Preferably, monoclonal antibodies are used in the diagnostic imagingmethods of the invention.

Cancer Immunotherapy and Cancer Vaccines

The invention provides various immunotherapeutic methods for treatingprostate cancer, including antibody therapy, in vivo vaccines, and exvivo immunotherapy methods, which utilize polynucleotides andpolypeptides corresponding to STEAP and STEAP antibodies. Thesetherapeutic applications are described further in the followingsubsections.

Applicants have accumulated strong and compelling evidence that STEAP-1is strongly expressed uniformly over the surface of glandular epithelialcells within prostate and prostate cancer cells. See, for details,immunohistochemical and Western blot analyses of STEAP-1 proteinexpression presented in Examples 3C and 3D as well as the STEAP-1 mRNAexpression profiles obtained from the Northern blot and RT-PCR generateddata presented in Examples 1 and 3A, B. In particular,immunohistochemical analysis results show that the surface of humanprostate epithelial cells (normal and cancer) appear to be uniformlycoated with STEAP-1. Biochemical analysis confirms the cell surfacelocalization of STEAP-1 initially suggested by its putative6-transmembrane primary structural elements and by the pericellularstaining plainly visualized by immunohistochemical staining.

STEAP-1 is uniformly expressed at high levels over the surface ofprostate glandular epithelia, an ideal situation for immunotherapeuticintervention strategies which target extracellular STEAP epitopes.Systemic administration of STEAP-immunoreactive compositions would beexpected to result in extensive contact of the composition with prostateepithelial cells via binding to STEAP-1 extracellular epitopes.Moreover, given the near absence of STEAP-1 protein expression in normalhuman tissues, there is ample reason to expect exquisite sensitivitywithout toxic, non-specific and/or non-target effects caused by thebinding of the immunotherapeutic composition to STEAP-1 on non-targetorgans and tissues.

In addition to the high level expression of STEAP-1 in prostate andprostate cancer cells, STEAP-1 appears to be substantiallyover-expressed in a variety of other human cancers, including bladder,colon, pancreatic and ovarian cancers. In particular, high level STEAP-1mRNA expression is detected in all tested prostate cancer tissues andcell lines, and in most of the pancreatic, colon, and bladder cancercell lines tested. High level expression of STEAP-1 is also observed insome ovarian cancer cell lines. Lower level expression is observed insome breast, testicular, and cervical cancer cell lines. Very high levelexpression is also detected in a Ewing sarcoma cell line. Applicantshave shown that cell surface STEAP-1 protein is expressed in bladder andcolon cancers, while there is no detectable cell surface (orintracellular) STEAP-1 protein in normal colon and low expression innormal bladder. Antibodies specifically reactive with extracellulardomains of STEAP-1 may be useful to treat these cancers systemically,either as toxin or therapeutic agent conjugates or as naked antibodiescapable of inhibiting cell proliferation or function.

STEAP-2 protein is also expressed in prostate cancer and may beexpressed in other cancers as well. STEAP-2 mRNA analysis by RT-PCR andNorthern blot show that expression is restricted to prostate in normaltissues, is also expressed in some prostate, pancreatic, colon,testicular, ovarian and other cancers. Therefore, antibodies reactivewith STEAP-2 may be useful in the treatment of prostate and othercancers. Similarly, although not yet characterized by applicants, theexpression of STEAP-3 and STEAP-4 (as well as other STEAPs) may beassociated with some cancers. Thus antibodies reactive with these STEAPfamily member proteins may also be useful therapeutically.

STEAP antibodies may be introduced into a patient such that the antibodybinds to STEAP on the cancer cells and mediates the destruction of thecells and the tumor and/or inhibits the growth of the cells or thetumor. Mechanisms by which such antibodies exert a therapeutic effectmay include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulating the physiologic function of STEAP, inhibitingligand binding or signal transduction pathways, modulating tumor celldifferentiation, altering tumor angiogenesis factor profiles, and/or byinducing apoptosis. STEAP antibodies conjugated to toxic or therapeuticagents may also be used therapeutically to deliver the toxic ortherapeutic agent directly to STEAP-bearing tumor cells.

Cancer immunotherapy using anti-STEAP antibodies may follow theteachings generated from various approaches which have been successfullyemployed with respect to other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit Rev Immunol18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186;Tsunenari et al., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyket al., 1992, Cancer Res 52: 2771-2776). B-cell lymphoma (Funakoshi etal., 1996. J Immunther Emphasis Tumor Immunol 19: 93-101), leukemia(Zhong et al., 1996, Leuk Res 20: 581-589), colorectal cancer (Moun etal., 1994, Cancer Res 54: 6160-6166); Velders et al., 1995, Cancer Res55: 4398-4403), and breast cancer (Shepard et al., 1991, J Clin Immunol11: 117-127).

Although STEAP antibody therapy may be useful for all stages of theforegoing cancers, antibody therapy may be particularly appropriate andin advanced or metastatic cancers. Combining the antibody therapy methodof the invention with a chemotherapeutic or radiation regimen may bepreferred in patients who have not received chemotherapeutic treatment,whereas treatment with the antibody therapy of the invention may beindicated for patients who have received one or more chemotherapy.Additionally, antibody therapy may also enable the use of reduceddosages of concomitant chemotherapy, particularly in patients that donot tolerate the toxicity of the chemotherapeutic agent very well.

It may be desirable for non-prostate cancer patients to be evaluated forthe presence and level of STEAP over-expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative STEAPimaging, or other techniques capable of reliably indicating the presenceand degree of STEAP overexpression. Immunohistochemical analysis oftumor biopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

Anti-STEAP monoclonal antibodies useful in treating prostate and othercancers include those which are capable of initiating a potent immuneresponse against the tumor and those which are capable of directcytotoxicity. In this regard, anti-STEAP mAbs may elicit tumor celllysis by either complement-mediated or antibody-dependent cellcytotoxicity (ADCC) mechanisms, both of which require an intact Fcportion of the immunoglobulin molecule for interaction with effectorcell Fc receptor sites or complement proteins. In addition, anti-STEAPmAbs which exert a direct biological effect on tumor growth are usefulin the practice of the invention. Potential mechanisms by which suchdirectly cytotoxic mAbs may act include inhibition of cell growth,modulation of cellular differentiation, modulation of tumor angiogenesisfactor profiles, and the induction of apoptosis. The mechanism by whicha particular anti-STEAP mAb exerts an anti-tumor effect may be evaluatedusing any number of in vitro assays designed to determine ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

The anti-tumor activity of a particular anti-STEAP mAb, or combinationof anti-STEAP mAbs, may be evaluated in vivo using a suitable animalmodel. For example, xenogenic prostate cancer models wherein humanprostate cancer explants or passaged xenograft tissues are introducedinto immune compromised animals, such as nude or SCID mice, areappropriate in relation to prostate cancer and have been described(Klein et al., 1997, Nature Medicine 3: 402-408). For Example, PCTPatent Application WO98/16628, Sawyers et al., published Apr. 23, 1998.describes various xenograft models of human prostate cancer capable ofrecapitulating the development of primary tumors, micrometastasis, andthe formation of osteoblastic metastases characteristic of late stagedisease. Efficacy may be predicted using assays which measure inhibitionof tumor formation, tumor regression or metastasis, and the like.

It should be noted that the use of murine or other non-human monoclonalantibodies, human/mouse chimeric mAbs may induce moderate to strongimmune responses in some patients. In the most severe cases, such animmune response may lead to the extensive formation of immune complexeswhich, potentially, can cause renal failure. Accordingly, preferredmonoclonal antibodies used in the practice of the therapeutic methods ofthe invention are those which are either fully human or humanized andwhich bind specifically to the target 20P1F12/TMPRSS2 antigen with highaffinity but exhibit low or no antigenicity in the patient.

The method of the invention contemplate the administration of singleanti-STEAP mAbs as well as combinations, or “cocktails, of differentmAbs. Such mAb cocktails may have certain advantages inasmuch as theycontain mAbs which exploit different effector mechanisms or combinedirectly cytotoxic mAbs with mAbs that rely on immune effectorfunctionality. Such mAbs in combination may exhibit synergistictherapeutic effects. In addition, the administration of anti-STEAP mAbsmay be combined with other therapeutic agents, including but not limitedto various chemotherapeutic agents, androgen-blockers, and immunemodulators (e.g., IL-2, GM-CSF). The anti-STEAP mAbs may be administeredin their “naked” or unconjugated form, or may have therapeutic agentsconjugated to them.

The anti-STEAP monoclonal antibodies used in the practice of the methodof the invention may be formulated into pharmaceutical compositionscomprising a carrier suitable for the desired delivery method. Suitablecarriers include any material which when combined with the anti-STEAPmAbs retains the anti-tumor function of the antibody and is non-reactivewith the subject's immune systems. Examples include, but are not limitedto, any of a number of standard pharmaceutical carriers such as sterilephosphate buffered saline solutions, bacteriostatic water, and the like.

The anti-STEAP antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumor, intradermal,and the like. The preferred route of administration is by intravenousinjection. A preferred formulation for intravenous injection comprisesthe anti-STEAP mAbs in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for injection,USP. The anti-STEAP mAb preparation may be lyophilized and stored as asterile powder, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Treatment will generally involve the repeated administration of theanti-STEAP antibody preparation via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. Doses in therange of 10-500 mg mAb per week may be effective and well tolerated.Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-STEAP mAb preparation may represent an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the mAb or mAbs used, the degree of STEAPoverexpression in the patient, the extent of circulating shed STEAPantigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

Optimally, patients should be evaluated for the level of circulatingshed STEAP antigen in serum in order to assist in the determination ofthe most effective dosing regimen and related factors. Such evaluationsmay also be used for monitoring purposes throughout therapy, and may beuseful to gauge therapeutic success in combination with evaluating otherparameters (such as serum PSA levels in prostate cancer therapy).

Cancer Vaccines

The invention further provides prostate cancer vaccines comprising aSTEAP protein or fragment thereof. The use of a tumor antigen in avaccine for generating humoral and cell-mediated immunity for use inanti-cancer therapy is well known in the art and has been employed inprostate cancer using human PSMA and rodent PAP immunogens (Hodge etal., 1995, Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol.159: 3113-3117). Such methods can be readily practiced by employing aSTEAP protein, or fragment thereof, or a STEAP-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the STEAP immunogen.

For example, viral gene delivery systems may be used to deliver aSTEAP-encoding nucleic acid molecule. Various viral gene deliverysystems which can be used in the practice of this aspect of theinvention include, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a STEAP protein or fragment thereof introduced into the patient(e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human STEAP cDNA may be employed. In anotherembodiment, STEAP nucleic acid molecules encoding specific cytotoxic Tlymphocyte (CTL) epitopes may be employed. CTL epitopes can bedetermined using specific algorithms (e.g., Epimer, Brown University) toidentify peptides within a STEAP protein which are capable of optimallybinding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present STEAP antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase 1 clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al.,1996, Prostate 29: 371-380). Dendritic cells can be used to presentSTEAP peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed withSTEAP peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete STEAP protein.Yet another embodiment involves engineering the overexpression of theSTEAP gene in dendritic cells using various implementing vectors knownin the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther.4: 17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribaset al., 1997, Cancer Res. 57: 2865-2869), and tumor-derived RNAtransfection (Ashley et al., 1997, J. Exp. Med. 186: 1177-1182).

Anti-idiotypic anti-STEAP antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga STEAP protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-STEAP antibodies that mimic an epitope on aSTEAP protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al.,1996, Cancer Immunol Immunother 43: 65-76). Such an anti-idiotypicantibody can be used in anti-idiotypic therapy as presently practicedwith other anti-idiotypic antibodies directed against tumor antigens.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing STEAP. Constructs comprising DNA encoding aSTEAP protein/immunogen and appropriate regulatory sequences may beinjected, directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded STEAP protein/immunogen. Expression of the STEAP proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against prostate cancer. Variousprophylactic and therapeutic genetic immunization techniques known inthe art may be used.

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe which is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a STEAP proteinor a STEAP gene or message, respectively. Where the kit utilizes nucleicacid hybridization to detect the target nucleic acid, the kit may alsohave containers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, fluorescent, orradionucleotide label.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples which follow, none of which are intendedto limit the scope of the invention.

Example 1 Isolation of cDNA Fragment of STEAP-1 Gene Materials andMethods Cell Lines and Human Tissues

All human cancer cell lines used in this study were obtained from theATCC. All cell lines were maintained in DMEM with 10% fetal calf serum.PrEC (primary prostate epithelial cells) were obtained from Cloneticsand were grown in PrEBM media supplemented with growth factors(Clonetics).

All human prostate cancer xenografts were originally provided by CharlesSawyers (UCLA) (Klein et al., 1997). LAPC-4 AD and LAPC-9 AD xenograftswere routinely passaged as small tissue chunks in recipient SCID males.LAPC-4 AI and LAPC-9 AI xenografts were derived as described previously(Klein et al., 1997) and were passaged in castrated males or in femaleSCID mice. A benign prostatic hyperplasia tissue sample waspatient-derived.

Human tissues for RNA and protein analyses were obtained from the HumanTissue Resource Center (HTRC) at the UCLA (Los Angeles, Calif.) and fromQualTek, Inc. (Santa Barbara, Calif.).

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

RSACDN (cDNA synthesis primer): (SEQ ID NO. 22) 5′TTTTGTACAAGCTT303′Adaptor 1: (SEQ ID NO. 23)5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT3′ (SEQ ID NO: 24)3′GGCCCGTCCA5′ Adaptor 2: (SEQ ID NO. 25)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT3′ (SEQ ID NO: 26)3′CGGCTCCA5′ PCR primer 1: (SEQ ID NO. 27) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO. 28) 5′TCGAGCGGCCGCCCGGGCAGGT3′Nested primer (NP)2: (SEQ ID NO. 29) 5′AGCGTGGTCGCGGCCGAGGT3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes which may be up-regulated in androgen dependentprostate cancer compared to benign prostatic hyperplasia.

Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester)and the BPH tissue (driver) were synthesized from 2 μg of poly(A)⁺ RNAisolated from xenograft and BPH tissue, as described above, usingCLONETECH'S PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotideRSACDN as primer. First- and second-strand synthesis were carried out asdescribed in the Kit's user manual protocol (CLONTECH Protocol No.PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with RsaI for 3 hrs. at 37° C. Digested cDNA was extracted withphenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA (BPH) was generated by combining in a 4 to 1 ratio Rsa Idigested BPH cDNA with digested cDNA from mouse liver, in order toensure that murine genes were subtracted from the tester cDNA (LAPC-4AD).

Tester cDNA (LAPC-4 AD) was generated by diluting 1 μl of Rsa I digestedLAPC-4 AD cDNA (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160ng) was then ligated to 2 μl of adaptor 1 and adaptor 2 (10 μM), inseparate ligation reactions, in a total volume of 10 μl at 16° C.overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) adaptor 1-and adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlayed with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl. 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12-18priming using the Gibco-BRL Superscript Preamplification system. Themanufacturers protocol was used and included an Incubation for 50 min at42° C. with reverse transcriptase followed by RNAse H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 μl with water prior to normalization. First strand cDNAs from 16different normal human tissues were obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:32) and 5′agccacacgcagctcattgtagaagg3′ (SEQ ID NO: 33) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1× PCR buffer(Clontech, 10 mM Tris-HCL, 1.5 mM MgCl.sub.2, 50 mM KCl, pH8.3) and 1×Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction wasremoved at 18, 20, and 22 cycles and used for agarose gelelectrophoresis. PCR was performed using an MJ Research thermal cyclerunder the following conditions: initial denaturation was at 94° C. for15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15 sec, 65° C.for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried outfor 2 min. After agarose gel electrophoresis, the band intensities ofthe 283 bp β-actin bands from multiple tissues were compared by visualinspection. Dilution factors for the first strand cDNAs were calculatedto result in equal β-actin band intensities in all tissues after 22cycles of PCR. Three rounds of normalization were required to achieveequal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 8P1 D4 gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs:

(SEQ ID NO. 30) 5′ ACT TTG TTG ATG ACC AGG ATT GGA 3′ (SEQ ID NO. 31) 5′CAG AAC TTC AGC ACA CAC AGG AAC 3′

Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results:

Several SSH experiments were conduced as described in the Materials andMethods, supra, and led to the isolation of numerous candidate genefragment clones. All candidate clones were sequenced and subjected tohomology analysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or Northern analysis.

One of the cDNA clones, designated 8P1 D4, was 436 bp in length andshowed homology to an EST sequence in the NCI-CGAP tumor gene database.The full length cDNA encoding the 8P1D4 gene was subsequently isolatedusing this cDNA and re-named STEAP-1 (Example 2, below). The 8P1D4 cDNAnucleotide sequence corresponds to nucleotide residues 150 through 585in the STEAP-1 cDNA sequence as shown in FIG. 1A. Another clone,designated 28P3E1, 561 bp in length showed homology to a number of ESTsequences in the NCI-CGAP tumor gene database or in other databases.Part of the 28P3E1 sequence (356 bp) is identical to an EST derived fromhuman fetal tissue. After the full length STEAP-1 cDNA was obtained andsequenced, it became apparent that this clone also corresponds toSTEAP-1 (more specifically, to residues 622 through the 3′ end of theSTEAP-1 nucleotide sequence as shown in FIG. 1A).

Differential expression analysis by RT-PCR using primers derived fromthe 8P1D4 cDNA clone showed that the 8P1D4 (STEAP-1) gene is expressedat approximately equal levels in normal prostate and the LAPC-4 andLAPC-9 xenografts (FIG. 2, panel A). Further RT-PCR expression analysisof first strand cDNAs from 16 normal tissues showed greatest levels of8P1D4 expression in prostate. Substantially lower level expression inseveral other normal tissues (i.e., colon, ovary, small intestine,spleen and testis) was detectable only at 30 cycles of amplification(FIG. 2, panels B and C).

Example 2 Isolation of Full Length STEAP-1 Encoding cDNA

The 436 bp 8P1D4 gene fragment (Example 1) was used to isolateadditional cDNAs encoding the 8P1D4/STEAP-1 gene. Briefly, a normalhuman prostate cDNA library (Clontech) was screened with a labeled probegenerated from the 436 bp 8P1D4 cDNA. One of the positive clones, clone10, is 1195 bp in length and encodes a 339 amino acid protein havingnucleotide and encoded amino acid sequences bearing no significanthomology to any known human genes or proteins (homology to a rat KidneyInjury Protein recently described in International ApplicationWO98/53071). The encoded protein contains at least 6 predictedtransmembrane motifs implying a cell surface orientation (see FIG. 1A,predicted transmembrane motifs underlined). These structural featuresled to the designation “STEAP”, for “Six Transmembrane EpithelialAntigen of the Prostate”. Subsequent identification of additional STEAPproteins led to the re-designation of the 8P1D4 gene product as“STEAP-1.” The STEAP-1 cDNA and encoded amino acid sequences are shownin FIG. 1A and correspond to SEQ ID NOS: 1 and 2, respectively. STEAP-1cDNA clone 10 has been deposited with the American Type CultureCollection (“ATCC”) (Mannassas, Va.) as plasmid 8P1D4 clone 10.1 on Aug.26, 1998 as ATCC Accession Number 98849. The STEAP-1 cDNA clone can beexcised therefrom using EcoRI/XbaI double digest (EcoRI at the 5′end,XbaI at the 3′end).

This deposit was made under the provisions of the Budapest Treaty onInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and for at least five (5) years afterthe most recent request for the furnishing of a sample of the depositreceived by the depository. The deposits will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures that all restrictionsimposed by the depositor on the availability to the public of thedeposited material will be irrevocably removed upon the granting of thepertinent U.S. patent, assures permanent and unrestricted availabilityof the progeny of the culture of the deposit to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 U.S.C.§122 and the Commissioner's rule pursuant thereto (including 37 C.F.R.§1.14 with particular reference to 886 OG 638).

Example 3 STEAP-1 Gene and Protein Expression Analysis

In order to begin to characterize the biological characteristics ofSTEAP-1, an extensive evaluation of STEAP-1 mRNA and STEAP-1 proteinexpression across a variety of human tissue specimens was undertaken.This evaluation included Northern blot, Western blot andimmunohistochemical analysis of STEAP-1 expression in a large number ofnormal human tissues, human prostate cancer xenografts and cell lines,and various other human cancer cell lines.

Example 3A

Northern Blot Analysis of STEAP-1 mRNA Expression in Normal HumanTissues

Initial analysis of STEAP-1 mRNA expression in normal human tissues wasconducted by Northern blotting two multiple tissue blots obtained fromClontech (Palo Alto, Calif.), comprising a total of 16 different normalhuman tissues, using labeled STEAP-1 clone 10 as a probe. RNA sampleswere quantitatively normalized with a β-actin probe. The results areshown in FIG. 3A. The highest expression level was detected in normalprostate, with an approximately 5-10 fold lower level of expressiondetected in colon and liver. These northern blots showed two transcriptsof approximately 1.4 kb and 4.0 kb, the former of which corresponds tothe full length STEAP-1 clone 10 cDNA, which encodes the entire STEAP-1open reading frame. The larger transcript was separately cloned as a3627 bp cDNA from a normal prostate library, the sequence of whichcontains a 2399 bp intron (FIG. 4).

This initial analysis was extended by using the STEAP-1 clone 10 probeto analyze an RNA dot blot matrix of 37 normal human tissues (Clontech,Palo Alto, Calif.; Human Master Blot™). The results are shown in FIG. 3Band show strong STEAP-1 expression only in prostate. Very low levelSTEAP-1 RNA expression was detected in liver, lung, trachea and fetalliver tissue, at perhaps a 5-fold lower level compared to prostate. Noexpression was detected in any of the remaining tissues. Based on theseanalyses, significant STEAP-1 expression appears to be prostate specificin normal tissues.

Example 3B

Northern Blot Analysis of STEAP-1 mRNA Expression in Prostate CancerXenografts and Cell Lines

To analyze STEAP-1 expression in human cancer tissues and cell lines,RNAs derived from human prostate cancer xenografts and an extensivepanel of prostate and non-prostate cancer cell lines were analyzed byNorthern blot using STEAP-1 cDNA clone 10 as probe. All RNA samples werequantitatively normalized by ethiduim bromide staining and subsequentanalysis with a labeled β-actin probe.

The results, presented in FIG. 5, show high level STEAP-1 expression inall the LAPC xenografts and all of the prostate cancer cell lines.Expression in the LAPC-9 xenografts was higher compared to the LAPC-4xenografts, with no significant difference observed betweenandrogen-dependent and androgen-independent sublines (FIG. 5A).Expression in the LAPC-4 xenografts was comparable to expression innormal prostate. Lower levels of expression were detected in PrEC cells(Clonetics), which represent the basal cell compartment of the prostate.Analysis of prostate cancer cell lines showed highest expression levelsin LNCaP, an androgen dependent prostate carcinoma cell line.Significant expression was also detected in the androgen-independentcell lines PC-3 and DU145. High levels of STEAP expression were alsodetected in LAPC-4 and LAPC-9 tumors that were grown within the tibia ofmice as a model of prostate cancer bone metastasis (FIG. 5B).

Significantly, very strong STEAP-1 expression was also detected in manyof the non-prostate human cancer cell lines analyzed (FIG. 5A).Particularly high level expression was observed in RD-ES cells, an Ewingsarcoma (EWS) derived cell line. Additionally, very high levelexpression was also detected in several of the colon cancer cell lines(e.g., CaCo-2, LoVo, T84 and Colo-205), bladder carcinoma cell lines(e.g., SCABER, UM-UC-3, TCCSUP and 5637), ovarian cancer cell lines(e.g., OV-1063 and SW 626) and pancreatic cancer cell lines (e.g., HPAC,Capan-1, PANC-1 and BxPC-3). These results, combined with the absence ofstrong expression in the corresponding normal tissues (FIG. 3), indicatethat STEAP-1 may be generally up-regulated in these types (as well asother types) of human cancers.

Example 3C Western Blot Analysis of STEAP-1 Protein Expression inProstate and Other Cancers

A 15 mer peptide corresponding to amino acid residues 14 through 28 ofthe STEAP-1 amino acid sequence as shown in FIG. 1A(WKMKPRRNLEEDDYL)(SEQ ID NO: 39) was synthesized and used to immunizesheep for the generation of sheep polyclonal antibodies towards theamino-terminus of the protein (anti-STEAP-1) as follows. The peptide wasconjugated to KLH (keyhole limpet hemocyanin). The sheep was initiallyimmunized with 400 μg of peptide in complete Freund's adjuvant. Theanimal was subsequently boosted every two weeks with 200 μg of peptidein incomplete Freund's adjuvant. Anti-STEAP antibody wasaffinity-purified from sheep serum using STEAP peptide coupled toAffi-Gel 10 (Bio Rad). Purified antibody is stored in phosphate-bufferedsaline with 0.1% sodium azide.

To test antibody specificity, the cDNA of STEAP-1 was cloned into aretroviral expression vector (pSRαtkneo, Muller et al., 1991, MCB11:1785). NIH 3T3 cells were infected with retroviruses encoding STEAP-1and were selected in G4I8 for 2 weeks. Western blot analysis of proteinextracts of infected and un-infected NIH 3T3 cells showed expression ofa protein with an apparent molecular weight of 36 kD only in theinfected cells (FIG. 6, lanes marked “313 STEAP” AND “3T3”).

The anti-STEAP-1 polyclonal antibody was used to probe Western blots ofcell lysates prepared from a variety of prostate cancer xenografttissues, prostate cancer cell lines and other non-prostate cancer celllines. Protein samples (20 μg each) were quantitatively normalized byprobing the blots with an anti-Grb-2 antibody.

The results are shown in FIG. 6. STEAP-1 protein was detected in all ofthe LAPC prostate cancer xenografts, all of the prostate cancer celllines, a primary prostate cancer specimen and its matched normalprostate control. Highest STEAP-1 protein expression was detected in theLAPC-9 xenograft and in LNCaP cells, in agreement with the Northern blotanalysis described immediately above. High level expression was alsoobserved in the bladder carcinoma cell line UM-UC-3. Expression in othercancer cell lines was also detectable (FIG. 6).

Example 3D Immunohistochemical Analysis of STEAP-1 Protein Expression inProstate Tumor Biopsy and Surgical Specimens

To determine the extent of STEAP-1 protein expression in clinicalmaterials, tissue sections were prepared from a variety of prostatecancer biopsies and surgical samples for immunohistochemical analysis.Tissues were fixed in 10% formalin, embedded in paraffin, and sectionedaccording to standard protocol. Formalin-fixed, paraffin-embeddedsections of LNCaP cells were used as a positive control. Sections werestained with an anti-STEAP-1 polyclonal antibody directed against aSTEAP-1 N-terminal epitope (as described immediately above). LNCaPsections were stained in the presence of an excess amount of the STEAP-1N-terminal peptide immunogen used to generate the polyclonal antibody(peptide 1) or a non-specific peptide derived from a distinct region ofthe STEAP-1 protein (peptide 2; YQQVQQNKEDAWIEH); (SEQ ID NO: 34)).

The results are shown in FIG. 8. LNCaP cells showed uniformly strongperi-cellular staining in all cells (FIG. 8B). Excess STEAP N-terminalpeptide (peptide 1) was able to competitively inhibit antibody staining(FIG. 8A), while peptide 2 had no effect (FIG. 8B). Similarly, uniformlystrong peri-cellular staining was seen in the LAPC-9 (FIG. 8F) andLAPC-4 prostate cancer xenografts (data not shown). These results areclear and suggest that the staining is STEAP specific. Moreover, theseresults visually localize STEAP to the plasma membrane, corroboratingthe biochemical findings presented in Example 4 below.

The results obtained with the various clinical specimens are shown inFIG. 8C (normal prostate tissue), FIG. 8D (grade 3 prostatic carcinoma),and FIG. 8E (grade 4 prostatic carcinoma), and are also included in thesummarized results shown in TABLE 1. Light to strong staining wasobserved in the glandular epithelia of all prostate cancer samplestested as well as in all samples derived from normal prostate or benigndisease. The signal appears to he strongest at the cell membrane of theepithelial cells, especially at the cell-cell junctions (FIGS. 8C, D andE) and is also inhibited with excess STEAP N-terminal peptide 1 (datanot shown). Some basal cell staining is also seen in normal prostate(FIG. 8 c), which is more apparent when examining atrophic glands (datanot shown). STEAP-1 seems to be expressed at all stages of prostatecancer since lower grades (FIG. 8D), higher grades (FIG. 8E) andmetastatic prostate cancer (represented by LAPC-9; FIG. 8F) all exhibitstrong staining.

Immunohistochemical staining of a large panel of normal non-prostatetissues showed no detectable STEAP-1 expression in 24 of 27 of thesenormal tissues (Table 1). Only three tissue samples showed some degreeof anti-STEAP-1 staining. In particular, normal bladder exhibited lowlevels of cell surface staining in the transitional epithelium (FIG.8G). Pancreas and pituitary showed low levels of cytoplasmic staining(Table I). It is unclear whether the observed cytoplasmic staining isspecific or is due to non-specific binding of the antibody, sinceNorthern blotting showed little to no STEAP-1 expression in pancreas(FIG. 3). Normal colon, which exhibited higher mRNA levels than pancreasby Northern blotting (FIG. 3), exhibited no detectable staining withanti-STEAP antibodies (FIG. 8H). These results indicate that cellsurface expression of STEAP-1 in normal tissues appears to be restrictedto prostate and bladder.

TABLE 1 IMMUNOHISTOCHEMICAL STAINING OF HUMAN TISSUES WITH ANTI-STEAP-1POLYCLONAL ANTIBODY STAINING INTENSITY TISSUE NONE cerebellum, cerebralcortex, spinal cord, heart, skeletal muscle, artery, thymus, spleen,bone marrow, lymph node, lung, colon, liver, stomach, kidney, testis,ovary, fallopian tubes, placenta, uterus, breast, adrenal gland, thyroidgland, skin, bladder (3/5) LIGHT TO bladder (2/5), pituitary gland(cytoplasmic), pancreas MODERATE (cytoplasmic), BPH (3/5), prostatecancer (3/10) STRONG prostate (2/2), BPH (2/5), prostate cancer** (7/10)*In cases where more than one sample is analyzed per tissue, the numbersin brackets indicates how many samples correspond to the stainingcategory/total analyzed. **Prostate cancer grades ranged from Gleasongrades 3 to 5.

Example 4 Biochemical Characterization of STEAP-1 Protein

To initially characterize the STEAP-1 protein, cDNA clone 10 (SEQ IDNO. 1) was cloned into the pcDNA 3.1 Myc-His plasmid (Invitrogen), whichencodes a 6His tag at the carboxyl-terminus, transfected into 293Tcells, and analyzed by flow cytometry using anti-His monoclonal antibody(His-probe, Santa Cruz) as well as the anti-STEAP-1 polyclonal antibodydescribed above. Staining of cells was performed on intact cells as wellas permeabilized cells. The results indicated that only permeabilizedcells stained with both antibodies, suggesting that both termini of theSTEAP-1 protein are localized intracellularly. It is therefore possiblethat one or more of the STEAP-1 protein termini are associated withintracellular organelles rather than the plasma membrane.

To determine whether STEAP-1 protein is expressed at the cell surface,intact STEAP-1-transfected 293T cells were labeled with a biotinylationreagent that does not enter live cells. STEAP-1 was thenimmunoprecipitated from cell extracts using the anti-His and anti-STEAPantibodies. SV40 large T antigen, an intracellular protein that isexpressed at high levels in 293T cells, and the endogenous cell surfacetransferrin receptor were immunoprecipitated as negative and positivecontrols, respectively. After immunoprecipitation, the proteins weretransferred to a membrane and visualized with horseradishperoxidase-conjugated streptavidin. The results of this analysis areshown in FIG. 7. Only the transferrin receptor (positive control) andSTEAP-1 were labeled with biotin, while the SV40 large T antigen(negative control) was not detectably labeled (FIG. 7A). Since only cellsurface proteins are labeled with this technique, it is clear from theseresults that STEAP-1 is a cell surface protein. Combined with theresults obtained from the flow cytometric analysis, it is clear thatSTEAP-1 is a cell surface protein with intracellular amino- andcarboxyl-termini.

Furthermore, the above results together with the STEAP-1 secondarystructural predictions, shows that STEAP-1 is a type IIIa membraneprotein with a molecular topology of six potential transmembranedomains, 3 extracellular loops, 2 intracellular loops and twointracellular termini. A schematic representation of STEAP-1 proteintopology relative to the cell membrane is shown in FIG. 1B.

In addition, prostate, bladder and colon cancer cells were directlyanalyzed for cell surface expression of STEAP-1 by biotinylationstudies. Briefly, biotinylated cell surface proteins were affinitypurified with streptavidin-gel and probed with the anti-STEAP-1polyclonal antibody described above. Western blotting of thestreptavidin purified proteins clearly show cell surface biotinylationof endogenous STEAP-1 in all prostate (LNCaP, PC-3, DU 145), bladder(UM-UC-3, TCCSUP) and colon cancer (LoVo, Colo) cells tested, as well asin NIH 3T3 cells infected with a STEAP-1 encoding retrovirus, but not innon-expressing NIH 3T3 cells used as a negative control (FIG. 7B). In afurther negative control, STEAP-1 protein was not detected instreptavidin precipitates from non-biotinylated STEAP expressing cells(FIG. 7B).

Example 5 Identification and Structural Analysis of STEAP-2 and OtherHuman STEAP Family Members

STEAP-1 has no homology to any known human genes. In an attempt toidentify additional genes that are homologous to STEAP-1, the proteinsequence of STEAP-1 was used as an electronic probe to identify familymembers in the public EST (expression sequence tag) database (dbest).Using the “tblastn” function in NCBI (National Center for BiotechnologyInformation), the dbest database was queried with the STEAP-1 proteinsequence. This analysis revealed additional putative STEAP-1 homologuesor STEAP family members, as further described below.

In addition, applicants cloning experiments also identified a STEAP-1related SSH cDNA fragment, clone 98P4B6. This clone was isolated fromSSH cloning using normal prostate cDNA as tester and LAPC-4 AD cDNA asdriver. A larger partial sequence of the 98P4B6 clone was subsequentlyisolated from a normal prostate library; this clone encodes an ORF of173 amino acids with close homology to the primary structure of STEAP-1,and thus was designated STEAP-2.

The STEAP-2 partial nucleotide and encoded ORF amino acid sequences areshown in FIG. 9. An amino acid alignment of the STEAP-1 and partialSTEAP-2 primary structures is shown in FIG. 11A. STEAP-1 and -2 share61% identity over their 171 amino acid residue overlap (FIG. 11A).Despite their homology, STEAP-1 and -2 show significantly divergentexpression patterns in normal and cancerous tissues and cells, and alsomap to distinct locations on opposite arms of human chromosome 7 (seeExamples 6 and 7 below).

Two ESTs identified by electronic probing with the STEAP-1 proteinsequence, AI139607 and R80991, encode ORFs bearing close homology to theSTEAP-1 and STEAP-2 sequences and thus appear to represent twoadditional STEAPs. Their nucleotide sequences are reproduced in FIG. 10and their encoded ORF STEAP-like amino acid sequences are shown in FIG.11B. The ORFs encoded by these ESTs are unique but show very clearstructural relationships to both STEAP-1 and STEAP-2, particularly inthe conserved transmembrane domains. Accordingly these ESTs appear tocorrespond to distinct STEAP family members and have thus beendesignated as STEAP-3 (corresponding to AI139607) and STEAP-4(corresponding to R80991).

An amino acid alignment of the complete STEAP-1 protein sequence withthe predicted partial STEAP-2, STEAP-3 and STEAP-4 amino acid sequencesis shown in FIG. 11B. This alignment shows a close structural similaritybetween all four STEAP family proteins, particularly in the predictedtransmembrane domains, even though only partial sequence information wasavailable for three of them. The STEAP-3 and STEAP-4 proteins appear tobe more closely related to STEAP-2 than to STEAP-1 or each other.Specifically, STEAP-3 shows 50% identity and 69% homology to STEAP-2,versus 37% identity and 63% homology to STEAP-1. STEAP-4 shows 56%identity and 87% homology to STEAP-2, versus 42% identity and 65%homology to STEAP-1. STEAP-3 and STEAP-4 are 38% identical and 57%homologous to each other. These figures are estimates based uponincomplete sequence information. However, these figures suggestconservation of at least some of the transmembrane domains, suggestingcommon topological characteristics if not functional characteristics.

Example 6 Expression Analysis of STEAP-2 and Other Human STEAP FamilyMembers Example 6A Tissue Specific Expression of STEAP Family Members inNormal Human Tissues

Expression analysis of STEAP family members in normal tissues wasperformed by RT-PCR. All STEAP family members appeared to exhibit tissuerestricted expression patterns. AI139607 expression is detected inplacenta and prostate after 25 cycles of amplification (FIG. 12). After30 cycles, AI139607 expression is also detected in other tissues. R80991expression is highest in normal liver, although expression is alsodetected in other tissues after 30 cycles of amplification (FIG. 13).Neither R80991, nor AI139607 expression was detected in the LAPCprostate cancer xenografts by RT-PCR.

RT-PCR analysis of STEAP-2 shows expression in all the LAPC prostatecancer xenografts and in normal prostate (FIG. 14A). Analysis of 8normal human tissues shows prostate-specific expression after 25 cyclesof amplification (FIG. 14B). Lower level expression in other tissues wasdetected only after 30 cycles of amplification. Northern blotting forSTEAP-2 shows a pattern of 2 transcripts (approximately 3 and 8 kb insize) expressed only in prostate (and at significantly lower levels inthe LAPC xenografts), with no detectable expression in any of the 15other normal human tissues analyzed (FIG. 15C). Thus, STEAP-2 expressionin normal human tissues appears to be highly prostate-specific.

Example 6B Expression of STEAP-2 in Various Cancer Cell Lines

The RT-PCR results above suggested that the different STEAP familymembers exhibit different tissue expression patterns. Interestingly.STEAP-2, which appears very prostate-specific, seems to be expressed atlower levels in the LAPC xenografts. This is in contrast to STEAP-1,which is highly expressed in both normal and malignant prostate tissue.

To better characterize this suggested difference in the STEAP-2 prostatecancer expression profile (relative to STEAP-1), Northern blotting wasperformed on RNA derived from the LAPC xenografts, as well as severalprostate and other cancer cell lines, using a STEAP-2 specific probe(labeled cDNA clone 98P4B6). The results are shown in FIG. 16 and can besummarized as follows. STEAP-2 is highly expressed in normal prostateand in some of the prostate cancer xenografts and cell lines. Moreparticularly, very strong expression was observed in the LAPC-9 ADxenograft and the LNCaP cells. Significantly attenuated or no expressionwas observed in the other prostate cancer xenografts and cell lines.Very strong expression was also evident in the Ewing Sarcoma cell lineRD-ES. Unlike STEAP-1, which is highly expressed in cancer cell linesderived from bladder, colon, pancreatic and ovarian tumors, STEAP-2showed low to non-detectable expression in these same cell lines(compare with FIG. 5). Interestingly, STEAP-2 was also non-detectable inPrEC cells, which are representative of the normal basal cellcompartment of the prostate. These results suggests that expression ofSTEAP-1 and STEAP-2 are differentially regulated. While STEAP-1 may be agene that is generally up-regulated in cancer, STEAP-2 may be a genethat is more restricted to normal prostate and prostate cancer.

Example 7 Chromosomal Localization of STEAP Genes

The chromosomal localization of STEAP-1 was determined using theGeneBridge 4 Human/Hamster radiation hybrid (RH) panel (Walter et al.,1994, Nat. Genetics 7:22) (Research Genetics, Huntsville, Ala.), whileSTEAP-2 and the STEAP homologues were mapped using the Stanford G3radiation hybrid panel (Stewart et al., 1997, Genome Res. 7:422).

The following PCR primers were used for STEAP-1:

8P1D4.1 5′ ACTTTGTTGATGACCAGGATTGGA 3′ (SEQ ID NO: 4) 8P1D4.2 5′CAGAACTTCAGCACACACAGGAAC 3′ (SEQ ID NO: 5)

The resulting STEAP-1 mapping vector for the 93 radiation hybrid panelDNAs(2100000201101010001000000101110101221000111001110110101000100010001-01001021000001111001010000), and the mapping program available at theinternet address for the Whitehead Institute for Biomedical researched,localized the STEAP-1 gene to chromosome 7p22.3, telomeric to D7S531.

The following PCR primers were used for 98P4B6/STEAP-2:

98P4B6.1 5′ GACTGAGCTGGAACTGGAATTTGT 3′ (SEQ ID NO: 17) 98P4B6.2 5′TTTGAGGAGACTTCATCTCACTGG 3′ (SEQ ID NO: 18)

The resulting vector(00000100100000000000000000000000100100000000001000100000000000001000010101010010011), and the mapping program available at theinternet address for the Stanford Human Genome Center, maps the 98P4B6(STEAP-2) gene to chromosome 7q21.

The following PCR primers were used for AI139607:

A1139607.1 5′ TTAGGACAACTTGATCACCAGCA 3′ (SEQ ID NO: 13) A1139607.2 5′TGTCCAGTCCAAACTGGGTTATTT 3′ (SEQ ID NO: 14)

The resulting vector(00000000100000000000000000001000100000200000001000100000001000000100010001010000010), and the mapping program available at theinternet address for the Stanford Human Genome Center, maps AI139607 tochromosome 7q21.

The following PCR primers were used for R80991:

R80991.3 5′ ACAAGAGCCACCTCTGGGTGAA 3′ (SEQ ID NO: 37) R80991.4 5′AGTTGAGCGAGTTTGCAATGGAC 3′ (SEQ ID NO: 38)

The resulting vector(00000000000200001020000000010000000000000000000010000000001000011100000001001000001), and the mapping program available atthe internet address for the Stanford Human Genome Center, maps R80991to chromosome 2q14-q21, near D2S2591.

In summary, the above results show that three of the putative humanSTEAP family members localize to chromosome 7, as is schematicallydepicted in FIG. 17. In particular, the STEAP-1 gene localizes to thefar telomeric region of the short arm of chromosome 7, at 7p22.3, whileSTEAP-2 and AI139607 localize to the long arm of chromosome 7, at 7q21(FIG. 17). R80991 maps to chromosome 2q14-q21.

Example 8 Identification of Intron-Exon Boundaries of STEAP-1

Genomic clones for STEAP-1 were identified by searching GenBank for BACclones containing STEAP-1 sequences, resulting in the identification ofaccession numbers AC004969 (PAC DJ1121E10) and AC005053 (BAC RG041D11).Using the sequences derived from the PAC and BAC clones for STEAP theintron-exon boundaries were defined (FIG. 18). A total of 4 exons and 3introns were identified within the coding region of the STEAP gene.Knowledge of the exact exon-intron structure of the STEAP-1 gene may beused for designing primers within intronic sequences which in turn maybe used for genomic amplification of exons. Such amplification permitssingle-stranded conformational polymorphism (SSCP) analysis to searchfor polymorphisms associated with cancer. Mutant or polymorphic exonsmay be sequenced and compared to wild type STEAP. Such analysis may beuseful to identify patients who are more susceptible to aggressiveprostate cancer, as well as other types of cancer, particularly colon,bladder, pancreatic, ovarian, cervical and testicular cancers.

Southern blot analysis shows that the STEAP-1 gene exists in severalspecies including mouse (FIG. 19). Therefore, a mouse BAC library (MouseES 129-V release 1, Genome Systems, FRAC-4431) was screened with thehuman cDNA for STEAP-1 (clone 10. Example 2). One positive clone, 12P11,was identified and confirmed by southern blotting (FIG. 20). Theintron-exon boundary information for human STEAP may be used to identifythe mouse STEAP-1 coding sequences.

The mouse STEAP-1 genomic clone may be used to study the biological roleof STEAP-1 during development and tumorigenesis. Specifically, the mousegenomic STEAP-1 clone may be inserted into a gene knock-out (K/O) vectorfor targeted disruption of the gene in mice, using methods generallyknown in the art. In addition, the role of STEAP in metabolic processesand epithelial cell function may be elucidated. Such K/O mice may becrossed with other prostate cancer mouse models, such as the TRAMP model(Greenberg et al., 1995, PNAS 92:3439), to determine whether STEAPinfluences the development and progression of more or less aggressiveand metastatic prostate cancers.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1. A method of delivering a cytotoxic agent, a therapeutic agent or adiagnostic agent to a cell that expresses a STEAP-1 protein (SEQ IDNO:2), comprising providing a cytotoxic agent, a therapeutic agent or adiagnostic agent conjugated to an antibody or antibody fragment that (a)specifically binds to a polypeptide comprising the amino acid sequenceof SEQ ID NO:2; (b) binds to an extracellular domain of the polypeptideof SEQ ID NO:2; (c) binds to a peptide selected from SEQ ID NO:19, SEQID NO:20, and SEQ ID NO:21; (d) binds to a cell surface epitope of thepolypeptide of SEQ ID NO:2 on NIH3T3 cells expressing the polypeptide;or (e) immunohistochemically stains 293T cells transfected with anexpression plasmid encoding and expressing a polypeptide having theamino acid sequence of SEQ ID NO:2 and does not immunohistochemicallystain untransfected 293T cells, wherein the antibody or antibodyfragment of (a)-(e) binds to the STEAP-1 protein (SEQ ID NO: 2), to forman antibody-agent or antibody fragment-agent conjugate; and exposing thecell to the antibody-agent or antibody fragment-agent conjugate.
 2. Themethod of claim 1, wherein the cytotoxic agent comprises a toxin.
 3. Themethod of claim 1 wherein the diagnostic agent comprises a detectablemarker.
 4. The method of claim 3, wherein the detectable marker is aradioisotope, a metal chelator, an enzyme, a substrate, a cofactor, aninhibitor, a fluorescent compound, a bioluminescent compound, a magneticparticle or a chemiluminescent compound.
 5. The method of claim 1,wherein the therapeutic agent comprises paclitaxel, an androgen blocker,or an immune modulator.
 6. The method of claim 1, wherein the antibodyis a monoclonal antibody.
 7. The method of claim 6, wherein themonoclonal antibody is a humanized antibody.
 8. The method of claim 1,wherein the antibody or antibody fragment are chimeric.
 9. The method ofclaim 1, wherein the antibody or antibody fragment thereof isrecombinantly produced.
 10. The method of claim 9, wherein therecombinant protein comprises the antigen binding region.
 11. The methodof claim 1, further comprising the administration of radiation,chemotherapy, or hormone ablation.
 12. The method of claim 1 wherein theantibody or antibody fragment is administered to a human patient who hasreceived one or more rounds of chemotherapy prior to the administrationof said antibody or antibody fragment.
 13. A method of inhibiting growthof cancer cells that express STEAP-1, the method comprising providing acytotoxic agent or a therapeutic agent conjugated to an antibody orantibody fragment that (a) specifically binds to a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; (b) binds to anextracellular domain of the polypeptide of SEQ ID NO:2; (c) binds to apeptide selected from SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21; (d)binds to a cell surface epitope of the polypeptide of SEQ ID NO:2 onNIH3T3 cells expressing the polypeptide; or (e) immunohistochemicallystains 293T cells transfected with an expression plasmid encoding andexpressing a polypeptide having the amino acid sequence of SEQ ID NO:2and does not immunohistochemically stain untransfected 293T cells,wherein the antibody or antibody fragment of (a)-(e) binds to theSTEAP-1 protein (SEQ ID NO: 2), to form an antibody-agent or antibodyfragment-agent conjugate; and exposing the cell to the antibody-agent orantibody fragment-agent conjugate.
 14. The method of claim 13, whereinthe cancer cells are from a cancer selected from the group consisting ofprostate, bladder, colon and ovarian cancer, and Ewing sarcoma.
 15. Themethod of claim 14, wherein the cancer cells are from malignantprostate.
 16. The method of claim 13, wherein the cytotoxic agentcomprises a toxin.
 17. The method of claim 13, wherein the therapeuticagent comprises paclitaxel, an androgen blocker, or an immune modulator.18. The method of claim 13, wherein the antibody is a monoclonalantibody.
 19. The method of claim 18, wherein the monoclonal antibody isa humanized antibody.
 20. The method of claim 13, wherein the antibodyor antibody fragment are chimeric.
 21. The method of claim 13, whereinthe antibody or antibody fragment thereof is recombinantly produced. 22.The method of claim 21, wherein the recombinant protein comprises theantigen binding region.
 23. The method of claim 13, further comprisingthe administration of radiation, chemotherapy, or hormone ablation. 24.The method of claim 13 wherein the antibody or antibody fragment isadministered to a human patient who has received one or more rounds ofchemotherapy prior to the administration of said antibody or antibodyfragment.